Caspase-3 Promotes Genetic Instability andCarcinogenesis
Article Caspase-3Promotes Genetic Instability and Carcinogenesis
Graphical Abstract
Highlights
d Cells exposed to ionizing radiation can surviv
e caspase-3
activation
d Caspase-3activation can caus
e persistent DNA strand
breaks in surviving cells
d Caspase-3promotes skin carcinogenesis induced by
DMBA+TPA treatment
d EndoG is a key downstream effector in causing DNA damage
mediated by caspase-3Authors
Xinjian Liu,Yujun He,...,Russell P.Hall, Chuan-Yuan Li
Correspondence
chuan.li@https://www.sodocs.net/doc/cf17399411.html,
In Brief
Activation of caspase-3is associated with the initiation of apoptosis.Liu et al. show that mammalian cells exposed to ionizing radiation or other stresses can survive with persistent caspase-3 activation,which in turn promotes genetic instability and oncogenic
transformation. Liu et al.,2015,Molecular Cell58,284–296
April16,2015a2015Elsevier Inc.
https://www.sodocs.net/doc/cf17399411.html,/10.1016/j.molcel.2015.03.003
Molecular Cell
Article
Caspase-3Promotes Genetic Instability
and Carcinogenesis
Xinjian Liu,1,6Yujun He,3,6Fang Li,1Qian Huang,4Takamitsu A.Kato,5Russell P.Hall,1and Chuan-Yuan Li1,2,*
1Department of Dermatology,Duke University Medical Center,Durham,NC27710,USA
2Department of Pharmacology and Cancer Biology,Duke University Medical Center,Durham,NC27710,USA
3Department of General Surgery,Daping Hospital,Third Military Medical University,Chongqing400042,China
4Cancer Center,First People’s Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai200080,China
5Department of Environmental and Radiological Health Sciences,Colorado State University,Fort Collins,CO80523,USA
6Co-?rst author
*Correspondence:chuan.li@https://www.sodocs.net/doc/cf17399411.html,
https://www.sodocs.net/doc/cf17399411.html,/10.1016/j.molcel.2015.03.003
SUMMARY
Apoptosis is typically considered an anti-oncogenic process since caspase activation can promote the elimination of genetically unstable or damaged cells. We report that a central effector of apoptosis, caspase-3,facilitates rather than suppresses chemi-cal-and radiation-induced genetic instability and carcinogenesis.We found that a signi?cant fraction of mammalian cells treated with ionizing radiation can survive despite caspase-3activation.Moreover, this sublethal activation of caspase-3promoted persistent DNA damage and oncogenic transforma-tion.In addition,chemically induced skin carcinogen-esis was signi?cantly reduced in mice genetically de?cient in caspase-3.Furthermore,attenuation of EndoG activity signi?cantly reduced radiation-induced DNA damage and oncogenic transformation, identifying EndoG as a downstream effector of cas-pase-3in this pathway.Our?ndings suggest that rather than acting as a broad inhibitor of carcinogen-esis,caspase-3activation may contribute to genome instability and play a pivotal role in tumor formation following damage.
INTRODUCTION
Apoptosis,or programmed cell death,is the most well-de?ned mode of cell death in multicellular organisms(Horvitz,2003;Hor-vitz et al.,1994;Yuan et al.,1993).A major physiological function of apoptosis is to get rid of damaged or unwanted cells in early development or to maintain somatic tissue homeostasis at later stages.As such,it is generally assumed that apoptosis is an anti-carcinogenic process due to its essential role in removing cells that have suffered DNA damage(Hanahan and Weinberg, 2000;Reed,1999).DNA damage and subsequent mutations in key oncogenes and tumor suppressor genes is a key process leading to cancer(Lengauer et al.,1997).Therefore,the current paradigm is that apoptosis is a barrier for carcinogenesis.For example,many tumor suppressor genes mutated in cancer often have apoptosis-promoting functions.Examples of these include p53(Lowe et al.,1994),PTEN(Weng et al.,2001),BAX(Wang et al.,1996),and INK4a/ARF(Kim et al.,2000).Mutations in these genes often allow damaged cells to survive when they should die.On the other hand,many oncogenes whose expressions are often enhanced in cancer cells possess anti-apoptotic functions.Examples of these include Bcl2(Cory and Adams, 2002),Bcl-x L(Cory and Adams,2002),Akt/PKB(Sabbatini and McCormick,1999),and mTOR(Panner et al.,2005). However,there is increasing recognition that the involvement of apoptosis in carcinogenesis may be more complicated.Some oncogenes appear to sensitize cells to apoptosis.For example, the apoptosis-promoting functions of c-Myc and E1A are well-documented in different studies(Debbas and White,1993; Evan et al.,1992;Fanidi et al.,1992;Rao et al.,1992).However, the role of apoptosis in carcinogenesis induced by these genes is not well-understood.For both c-Myc and E1A,their pro-apoptotic activities appear coupled to their hyperproliferative activities.Thus,it was suggested that apoptosis acts as a fail-safe mechanism to limit the consequences of aberrant mitogenic signaling(Lowe and Lin,2000).There was also a suggestion that oncogene-mediated excessive apoptosis might create a selec-tion pressure to overcome apoptosis,thus allowing cancer cells to become more malignant(Lowe and Lin,2000).However, these hypotheses have not been experimentally evaluated. Evidence is emerging that some factors in the apoptotic ma-chinery may play facilitative roles in carcinogenesis.An example is Fas Ligand(CD95),which is a major player in mediating the extrinsic pathway of cellular apoptosis.Genetic knockout of Fas Ligand attenuated tumorigenesis in mice instead of promot-ing it.Fas Ligand was shown to promote carcinogenesis by acti-vating downstream c-Jun and JNK pathway(Chen et al.,2010). Fas Ligand has also been shown to promote cancer cell inva-siveness and metastasis by interacting and activating c-Met oncogene(Lin et al.,2012).Another recent study showed that higher levels of activated caspase-3in head and neck cancer or breast cancer were correlated with increased post therapy tumor recurrence or patient death,contrary to conventional wisdom(Huang et al.,2011).
In this study,we designed experiments to examine the counter-intuitive hypothesis that caspase-3facilitates carcinogenesis
by
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inducing genetic instability in cells that survived cytotoxic stress. We provided strong evidence that activated caspase-3could indeed promote oncogenic transformation in human cells and in mice by inducing persistent genetic instability through its down-stream effector,endonuclease G,whose normal function is to fragment genomic DNA in apoptotic cells.
RESULTS
Non-lethal Activation of Caspase-3in Irradiated Cells Caspases are crucial enzymes in apoptosis through which damaged or unwanted cells systematically destroy their own infrastructure to commit suicide.Activation of caspases in cells exposed to cytotoxic stress(e.g.,radiation)is usually consid-ered an irreversible process that will result in the elimination of host cells.Thus,it has been generally assumed that all cells with apoptotic caspase activation will die.However,data in this respect are lacking for mammalian cells.We decided to carry out a detailed analysis of the relationship between cas-pase-3activation and the fate of host cells,because caspase-3is involved at the end stage of apoptosis(Nicholson et al., 1995;Taylor et al.,2008).To do this,we used a non-invasive caspase-3reporter recently developed in our laboratory(Fig-ure1A)(Huang et al.,2011;Li et al.,2010).In this reporter,the EGFP gene is fused with a?re?y luciferase gene reporter through a?exible linker.The fusion protein is further linked up with a polyubiquitin domain,which renders the fusion reporter protein very unstable because it is recognized by the protea-some system as an ubiquitylated protein and rapidly degraded. In between the EGFP-luc and polyubiquitin domains,a cas-pase-3cleavage site is inserted.Under normal circumstances steady-state reporter protein levels will be very low in host cells.However,when caspase-3is activated,the polyubiquitin domain will be cleaved off and the EGFP-Luc fusion protein will be stabilized.
To study the fate of cells with caspase-3activation,we trans-duced the Casp3EGFP-Luc reporter gene into the MCF10A hu-man mammary epithelial cells.We then subjected the cells to a moderate dose(0.5grays[Gy])of high energy(600million elec-tron volts per micrometer[MeV/m])56Fe ion irradiation.High en-ergy particle radiation has been shown to be more potent in inducing DNA damage and carcinogenesis compared with lower energy radiation(X-rays and g-rays)(Durante and Cucinotta, 2008).We show that radiation-induced signi?cant caspase-3 reporter activation in a persistent manner,with some cells ex-pressing high EGFP at2weeks post irradiation.To characterize reporter activation,the irradiated cells were sorted by use of?uo-rescence-activated cell sorting(FACS)according to their cellular GFP levels.Those with high GFP expression were separated from those with low GFP expression(Figure1B).Interestingly, many cells with high EGFP levels appeared normal morphologi-cally.This was despite robust cleaved caspase-3cleavage and cytochrome C release status in irradiated cells in general(Fig-ure S1A;see Table S1for a list of antibodies used in this study) and in cells with high EGFP levels(Figure1C).Those data,there-fore,contradict with the currently established view that damaged cells with caspase-3activation are destined to die.We further carried out experiments to observe the growth of the cells with high Casp3EGFP reporter activities.Our data suggest that both the EGFP-hi and EGFP-low groups of cells maintained continuous cellular growth and irradiated cells with high or low GFP expression grew only slightly slower than untreated parental cells(Figure S1B).Because caspase-3activation is often accompanied by cytochrome C leakage in apoptotic cells, we carried out immuno?uorescence analysis of cytochrome C in parental and Casp3EGFP-high cells.Our data(Figures S1C and S1D)showed that many irradiated GFP-hi cells showed a distinct cytochrome C staining pattern with a small,but signi?cant,per-centage of cytochrome C located in the extra-mitochondrial re-gions(Figure S1C,lower panels).In contrast,the vast majority of non-irradiated cells showed only mitochondrial cytochrome C staining(Figure S1C,top panels).Quantitatively,the per-centage of GFP-hi cells with the‘‘leaky’’extra-mitochondrial cytochrome C staining pattern was about23%at14days after irradiation.This is compared with about0.5%with the leaky pattern in the non-irradiated cells(Figure S1D).The persistence of cells with cytochrome C leakage suggests that the leakage was at levels that did not lead to cell death.An analysis of mito-chondrial membrane potential by use of the tetramethylrhod-amine,ethyl ester(TMRE)-?ow cytometry assay(Figure S1E) indicated that compared with parental MCF10A cells,those with high Casp3EGFP reporter activities showed higher degrees of mitochondrial membrane depolarization,indicating persistent and incomplete mitochondrial membrane leakage.Those data were also consistent with the leaky cytochrome C pattern shown in Figures S1C and S1D.
To further characterize the fate of individual cells with cas-pase-3activation,MCF10A cells transduced with caspase-3 reporters were irradiated with different doses of radiation and analyzed through?ow cytometry(Figure1D).The cells were then sorted through eight different ranges according to cas-pase-3reporter activation levels(Figures1D and1E).Higher ra-diation doses increased the fraction of cells with higher levels of caspase-3activation(Figures1D and1F).Cells from different ranges were then sorted one cell/well into different wells of96-well plates.EGFP expression in the wells was further con?rmed though?uorescence microscopy(data not shown). All the individual wells examined showed positive identi?cation of one cell/well.In addition,EGFP expression was seen clearly in cells from M4–M8ranges.Colony formation from the individ-ually plated cells with different GFP expression status(M1–M8) was then analyzed after2weeks in the96-well plates.As ex-pected,there was a clear radiation dose-dependent decrease in colony forming abilities(Figure1G).However,the relationship between caspase-3activation and colony forming abilities was more complicated.It appeared that cells could tolerate a wide range of caspase-3activation levels(Figure1G,M1–M4),espe-cially at lower doses of radiation.At a moderate radiation dose(3Gy),the relationship between caspase-3activation and clonogenic survival become linear,with M4as the threshold. At higher levels(>3Gy),most of the cells could not form colonies irrespective of caspase-3reporter activities,consistent with the ability of radiation to kill cells in an apoptosis-independent manner.Our data thus clearly established that radiation could activate caspase-3in a non-lethal manner at moderate radiation doses(%6Gy).
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Figure1.Non-lethal Activation of Caspase-3in MCF10A Cells Exposed to Ionizing Radiation
(A)Diagram of the caspase-3reporter gene.(Ub)9,a nine-ubiquitin polyubiqutin domain that serves as the proteasome recognition signal that causes the rapid degradation of the reporter protein,which consists of EGFP and?re?y luciferase linked by a?exible linker.
(B)Irradiated(0.5Gy600MeV56Fe ions)MCF10A cells with low(top panels)and high(lower panels)caspase-3reporter activities after separation by a FACS sorter based on Luc-GFP expression levels7days after cellular exposure to radiation.
(C)Western blot analysis of caspase-3cleavage and activation in cells with high and low Casp3EGFP reporter activities14days after irradiation.Cytochrome C was probed using cytosolic extracts,while other proteins were probed with whole cell lysates.
(D)Flow cytometry pro?les of MCF10A-CASP3EGFP reporter activities in cells exposed to different doses of X-rays.Cells were analyzed4days after irradiation. Cells were ranged into eight different groups for colony forming assays according to their?uorescence intensity levels(M1–M8).
(E)The mean(geometric)GFP?uorescence intensities of ranged MCF10A cells.
(F)Distribution of MCF10A cells in each range after different irradiation doses.For each radiation dose,M1+M2+.+M8=100%.
(G)Colony forming abilities of cells with different Casp3EGFP reporter activities.Cells from varying levels of reporter activities(M1–M8)were?ow-sorted into individual wells of96-well plates at one cell/well.At two weeks later,the numbers of MCF10A colonies on each96-well plate were counted and plotted. Error bars in(E)–(G)represent SD.All values are derived from the average of three independent experiments.See also Figure S1.
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A Key Facilitative Role of Caspase-3in Radiation-Induced DNA Damage Foci Formation in Surviving Cells Our observation of non-lethal caspase-3activation in MCF10A cells points to an important question:what happens to the genome of the cells that survive caspase-3activation?.This question is important since caspase-3activation in those cells could lead to downstream nuclease activation and DNA fragmen-tation,similar to what occurs in apoptotic cells,which can poten-tially wreak havoc in the genomes of cells experiencing ‘‘abortive apoptosis’’.To answer this question,we carried out experiments to examine DNA damage in cells that have survived caspase-3activation.Our data show that radiation exposure induced signif-icantly higher levels of g H2AX foci,an indication of DNA double strand breaks (DSBs)(Rogakou et al.,1998),when compared with sham-irradiated cells (Figures 2A and 2B,left side)at 14days after irradiation.There was a dose-dependent increase in the number of g H2AX foci in irradiated cells (Figure S1F).Furthermore,cells with higher levels of Casp3EGFP reporter acti-vation had signi?cantly more g H2AX foci,when compared with those with low levels of reporter activation (Figure 2B,right side).Importantly,higher numbers of g H2AX foci persist in the irradiated MCF10A cells for more than three months (Figure S1G).To determine if activation of caspase-3plays a causative role in the observed DNA strand breaks,we established
MCF10A
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E
F
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Figure 2.A Key Role for Activated Caspase-3in Radiation-Induced Foci Formation
(A)Typical micrographs of g H2AX foci in non-irradiated (0Gy)and irradiated (0.5Gy 56Fe ions)MCF10A cells.
(B)The average number of g H2AX foci in Casp3reporter-transduced MCF10A cells 14days after being exposed to 0.5Gy of 56Fe ions.The left two bars show the numbers for non-irradiated versus 0.5Gy irradiated cells,while the right two bars show the numbers for irradiated MCF10A cells with high and low caspase-3reporter activities based on GFP expression levels.
(C)The effect of caspase-3expression attenuation on g H2AX foci formation.MCF10A cells transduced with an shRNA gene against the CASP3gene (shCASP3)or control (shControl )were evaluated for g H2AX foci formation at 10days after 0.5Gy or sham irradiation.
(D)The effect of caspase-3inhibition on radiation-induced g H2AX foci formation in MCF10A cells.Cells transduced with a control lentiviral vector or those transduced with a dominant negative caspase-3gene (CASP3DN )were evaluated for g H2AX foci formation with sham or 0.5Gy of irradiation at 14days after irradiation.
(E)Typical micrographs of g H2AX foci in non-irradiated (0Gy)and irradiated (3Gy X-rays)MEF cells at 4days after exposure.
(F)The numbers of g H2AX foci in irradiated caspase-3de?ciency MEFs were signi?cantly lower than those of caspase-3pro?ciency MEFs at 4days after irradiation.
Error bars in (B)–(D)and (F)represent SEM.All values are derived from the average of three independent experiments.See also Figures S1–S3.
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cells that express a small hairpin RNA(shRNA)gene against the caspase-3gene(see Figure S2A,top panels for con?rmation of knockdown).In those cells,the numbers of g H2AX foci were signi?cantly reduced after exposure to56Fe ions radiation when compared with those transduced with a control shRNA gene(Figure2C).
The causative role of caspases-3was further con?rmed by use of MCF10A cells transduced with a dominant-negative version of caspase-3(Casp3DN,Figure S2A,lower panels).The dominant-negative version of the caspase-3has one single-base pair mutation that inactivates its cleavage activity(Stennicke and Sal-vesen,1997).As shown in Figure2D,the number of g H2AX foci was signi?cantly lower in irradiated MCF10A cells expressing Casp3DN at14days after irradiation.
In addition to the above data implicating caspase-3in medi-ating radiation-induced DNA damage in human cells,we also examined radiation-induced g H2AX foci formation using wild-type(WT)and Casp3KO mouse embryonic?broblast(MEF)cells. We observed signi?cantly more attenuated radiation-induced foci formation in Casp3KO,than in WT MEF cells(Figures2E and2F).Thus,our data support a key role for caspase-3in medi-ating radiation-induced g H2AX foci formation in murine cells as well.
Previous studies have shown that caspase-3and caspase-7 share a similar cleavage site,but are functionally distinct in their in?uences on host mice when each was genetically ablated(La-khani et al.,2006;Walsh et al.,2008).In order to compare the relative contribution of caspase-3and caspase-7,we also exam-ined g H2AX foci formation in WT,Casp3KO,Casp7KO,and cas-pase-3and caspase-7double knockout(DKO)cells(Figure S2B). Our data show that Casp3knockout had a more pronounced ef-fect than Casp7knockout.In addition,DKO cells did not exhibit less radiation-induced DNA damage than caspase-3knockout cells alone,indicating that caspase-3and caspase-7have mostly overlapping functions in terms of DNA damage,with cas-pase-3playing a much more dominant role.
We next examined dynamic53BP1foci formation in irradiated cells.53BP1is a protein shown to be actively involved in DSB repair(Dimitrova et al.,2008;Wang et al.,2002).It forms foci with many other DNA DSB repair proteins such as g H2AX at sites of https://www.sodocs.net/doc/cf17399411.html,ing a non-invasive red?uorescent protein(mCherry) based53BP1foci reporter(Dimitrova et al.,2008),we monitored 53BP1foci formation in the nuclei of irradiated WT and Casp3-de?ceint MEF cells.Our results show a signi?cantly higher frac-tion of cells with53BP1foci in irradiated WT MEF cells when compared with sham irradiated controls(Figures S2C and S2D).In addition,de?ciency in caspase-3caused a signi?cant reduction in53BP1foci(Figure S2D),similar to g H2AX foci.By time-lapse video,we were also able to observe dynamic and persistent53BP1foci formation in irradiated MCF10A cells and their progeny(Figure S2E).
Another important question is whether the observed caspase-3-dependent foci formation requires initial cellular exposure to ionizing radiation.In order to examine this issue,we transduced an arti?cially inducible,dimerizable Casp3gene(iCasp3)into MCF10A cells.The iCasp3gene is engineered by fusing cas-pase-3gene with FK506-binding sites(FKBPs)so that the engi-neered iCasp3protein can dimerize and be activated when exposed to an FK506analog in the absence of upstream apoptotic signaling(Fujioka et al.,2011;MacCorkle et al., 1998).We show that the addition of the chemical dimerizer (AP20187,an FK506analog)was suf?cient to induce caspase-3 activation and g H2AX foci formation(Figures S3A–S3C).These results indicate that caspase-3activation alone is suf?cient to induce DNA damage in host cells.
In another experiment,we evaluated g H2AX foci formation in WT and Casp3KO MEF cells exposed to staurosporine,an apoptosis inducer not known to cause direct DNA damage like ionizing radiation.Our results showed that staurosporine was able to induce g H2AX foci formation3days after WT MEF cells were exposed to it.However,the induction was almost abol-ished in Casp3KO cells(Figures S3D and S3E).These results provide further support for activated caspase-3in inducing DNA damage.
Evidence for Involvement of Caspase-3in
Radiation-Induced Persistent DNA Strand Breaks
as Determined by Use of the Comet Assay
We next determined the roles of caspase-3in radiation-induced persistent DNA damage in MCF10A cells using the comet assay, another approach to measure DNA strand breaks(Olive et al., 1990;Ostling and Johanson,1984;Singh et al.,1988).Our experiments show that in MCF10A cells exposed to56Fe ions irradiation,the‘‘comet tails’’’of the cells whose sizes re?ect the extent of DNA damage in host cells,were signi?cantly bigger than those of the control cells at14days after irradiation(Figures 3A and3B),indicating signi?cantly higher levels of persistent DNA strand breaks in the irradiated cells.Furthermore,those cells with higher levels of caspase-3activities possessed signif-icantly larger DNA tails when examined by use of the comet assay(Figure3B).In MCF10A cells transduced with an shRNA gene against caspase-3,comet assay analyses detected signif-icantly reduced levels of radiation-induced DNA damage when compared with WT cells at10days after irradiation(Figure3C). Similar results were also obtained with cells transduced with CASP3DN(Figure3D).Thus,results obtained with comet assay are very consistent with results obtained with the g H2AX assay (Figure2).
In addition to immortalized MCF10A cells,we examined the roles of caspase-3in56Fe ions radiation-induced comet tails in IMR90cells,a primary human?broblast cell strain.Our data show that caspase-3attenuation signi?cantly downregulated ra-diation-induced DNA damage in terms of comet assays in those cells as well(Figure3E).The role of caspase-3in radiation-induced DNA damage in IMR90cells was further examined by use of the g H2AX foci assay(Figure S3F)and chromosome ab-erration assay(Figure S3G).Our results show that shRNA against caspase-3(shCasp3)-mediated caspase-3attenuation reduced 56Fe ions induced DNA damage in both assays in IMR90cells. We also carried out experiments to determine if the p53pro-tein,the‘‘guardian of the genome’’,plays any role in radiation-induced,persistent DNA damage as measured by g H2AX foci formation.We show that in MCF10A cells,radiation-induced p53and its downstream p21protein expression at24hr post exposure,similar to IMR90cells(Figure S4A).By10days post irradiation,both p53and p21expression mostly went back to
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control levels (Figure S4A).In MCF10A cells transduced with TP53DN ,a dominant-negative version of TP53(Kendall et al.,2005),radiation-induced p21induction is signi?cantly reduced compared with parental MCF10cells.However,when g H2AX foci formation was quanti?ed in these cells at different time points post irradiation,we showed that no statistical difference exists between these two cell populations (Figure S4B),indi-cating the p53did not play a signi?cant role.
Evidence Implicating an Important Role for Caspase-3Activation in Radiation Induced Chromosome Aberrations
We conducted chromosome aberration analyses to further deter-mine the roles of caspase-3in radiation-induced chromosome instability.Chromosome instability is key characteristic of cancer cells (Lengauer et al.,1997,1998).We carried out chromosome aberration analysis in MCF10A cells.Our results show that inhibi-tion of caspase-3by use of CASP3DN signi?cantly reduced radiation-induced chromosomal aberrations in MCF10A cells (Figures 4A and S4C–S4F ;Table S2).
We also assessed radiation-induced chromosome aberrations in vivo in WT or Casp3de?cient (Casp3KO )C57BL/6mice (Kuida et al.,1996).Radiation-induced a signi?cant amount of chromo-some aberrations in both WT and Casp3KO bone marrow cells (Figure 4B;also see Table S3).On the other hand,bone marrow cells in the caspase-3knockout mice showed signi?-cantly less chromosome aberration after exposure to radiation when compared with WT
mice.
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Figure 3.A Key Role for Caspase-3in Radiation-Induced DNA Damage as Determined by the Comet Assay
(A)Typical examples of control and irradiated cells when run through electrophoresis during the comet assay.
(B)The fraction of DNA in the comet tail in caspase-3reporter-transduced MCF10A cells exposed to 0.5Gy of 56Fe ions 14days after 56Fe ions irradiation.The left two bars show fractions for non-irradiated versus 0.5Gy irradiated cells,while the right two bars show the fractions for irradiated MCF10A cells with high and low caspase-3reporter activities based on GFP expression levels.
(C)The effect of caspase-3expression attenuation on the fraction of DNA in the comet tails.MCF10A cells transduced with an shCASP3or control (shControl )were evaluated for the fraction of DNA in the comet tails at 10days after 0.5Gy or sham irradiation.
(D)The effect of caspase-3inhibition on radiation-induced DNA strand breaks as quanti?ed by the amount of cellular DNA in the comet tail in MCF10A cells.Cells transduced with a control lentiviral vector or those transduced with a dominant-negative caspase-3gene (CASP3DN )were evaluated for the amount of DNA in the comet tails with sham or 0.5Gy of irradiation at 14days after irradiation.
(E)The effect of caspase-3attenuation on the fraction of DNA in the comet tails in IMR90cells.IMR90cells transduced with an shCASP3or control (shControl )were evaluated for the fraction of DNA in the comet tails at 10days after 0.5Gy or sham irradiation.
Error bars in (B)–(E)represent SD.All values are derived from the average of three different samples within one experimental group.For each group,at least 50cells from randomly chosen ?elds were scored by use of the Image J software (NIH)for DNA distribution.A two-sided Student’s t test was used to calculate the p values.See also Figures S3and S4.
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In additional experiments,we examined radiation-induced chromosome translocations in mouse bone marrow cells by use of whole chromosome painting to evaluate the frequencies of chromosomes 1and 2translocations.Bone marrow cells in the caspase-3knockout mice showed signi?cantly less chromo-some 1and 2translocations after exposure to radiation when compared with WT mice (Figures 4C and 4D).
In further experiments,we examined the relative contribution of caspase-3and caspase-7in radiation-induced chromosome aberrations by use of mouse embryonic ?broblasts from WT,Casp3KO ,and Casp7KO cells.Our results (Figure 4E)show that both Casp3KO and Casp7KO cells exhibit signi?cantly reduced,radiation-induced chromosome aberrations with cas-pase-3playing a more prominent role.On the other hand,the caspase-3and caspase-7DKO MEF cells did not show addi-tional reduction in chromosome aberrations,indicating that Casp3plays a more dominant role in facilitating radiation-induced chromosome aberrations.
Caspase-3Plays a Facilitating Role in Oncogenic Transformation of MCF10A Cells Induced by Ionizing Radiation
Our results so far clearly established a causative role for caspase-3in radiation-induced genetic instability at both the DNA and chromosome levels.We next explored the relationship between non-lethal caspase-3activation and oncogenic transformation because genetic instability has been closely linked with carcino-genesis.We ?rst use FACS to sort out cells with low or high cas-pase-3activities.About 20%of the cells showed signi?cantly increased caspase-3reporter activities after 0.5Gy 600MeV 56
Fe ions irradiation (data not shown).Those cells were then
cultured for 2weeks and then plated into soft agar.The ability to grow in an anchorage-independent manner in soft agar is a hallmark of transformed cells (Cifone and Fidler,1980;Weinberg,2007).Our data show that MCF10A cells exposed to 56Fe ions readily formed colonies in soft agar (Figures 5A–5C,also see S5A–S5C for actual photos of soft agar colonies).Interestingly,those with high Casp3EGFP activities formed soft agar col-onies at signi?cantly higher frequencies than those with low Casp3EGFP caspase-3activities (Figure 5A).Those data suggest that higher caspase-3activities correlated with signi?cantly higher frequencies of oncogenic transformation despite minimal effect of caspase-3activation on the proliferation of the cells in culture (Figure S1B).We further examined if a causative relation-ship exists between caspase-3activation and soft agar growth in irradiated MCF10A cells by use of shCasp3and casp3DN to block caspase-3activities.We observed signi?cantly lower rates of soft agar colony formation in irradiated MCF10A-shCASP3cells (Figure 5B).In further experiments,we showed that cells with Casp3DN expression also had less radiation-induced soft agar colony formation (Figure 5C).Our results thus con?rmed a signi?cant facilitative role for capases-3in radiation-induced oncogenic transformation of MCF10A cells.We further attemp-ted to con?rm the tumorigenic nature of the irradiated cells in nude mice.Non-irradiated parental MCF10A cells did not form any tumors in nude mice (0/10)12weeks post subcutaneous in-jection into nude nice.On the other hand,irradiated (0.5Gy 56Fe ions)MCF10A cells readily formed tumors in nude mice (Figures 5D–5F).In fact,six out of ten mice injected with irradiated MCF10A cells formed tumors (Figure 5G).In contrast,neither control nor irradiated MCF10A-shCASP3or MCF10A-CaspDN cells formed any tumors (0/10for each of the two
experimental
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E Figure 4.A Key Role for Caspase-3Activ-ities in Radiation-Induced Chromosome Aberrations
(A)The effect of caspase-3inhibition on radiation-induced chromosome aberrations in MCF10A cells.Cells transduced with a control lentiviral vector or a dominant-negative caspase-3gene (CASP3DN )were evaluated for chromosome ab-errations with sham or 0.5Gy of 600MeV 56Fe ions irradiation at 14days after irradiation.
(B)Radiation-induced chromosome aberrations in WT or CASP3gene knockout (CASP3KO )C57BL/6mice.Results were obtained from bone marrow cells harvested from irradiated mice 3days after 3Gy X-ray irradiation.
(C)Whole chromosome painting results for chro-mosomes 1(FITC)and 2(rhodamine).A cellular chromosome spread showing a translocation of part of chromosome 2(arrow in red)in irradiated WT C57BL/6mice.
(D)Quantitative summary of radiation-induced chromosome 1and 2translocations in WT or
CASP3gene knockout (CASP3KO )C57BL/6mice.Results were obtained from bone marrow cells harvested from irradiated mice 3days after.No translocations were identi?ed in cells from either WT or CASP3KO mice without irradiation.
(E)Radiation-induced chromosomal aberrations in WT ,Casp3KO ,Casp7KO ,and Casp3KOCasp7KO (DKO )mouse embryonic ?broblast cells 5days post irradiation.
Error bars in (A),(B),(D),and (E)represent SD.All values are derived from the average of three separate experiments.In each experiment,150cells for each cell type were counted without knowledge of the cell types to enumerate chromosome aberrations.A two-tailed Student’s t test was used to calculate the p values.See also Figure S4and Tables S2and S3.
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groups,Figure 5F).Similar results were also obtained for MCF10A-CASP3DN cells.None of the mice injected with either non-irradiated or irradiated cells formed any tumors in 12weeks of observation (0/10for each group,Figure 5F).These data indi-cate that caspase-3activities are required for tumorigenicity in 56
Fe ions irradiated MCF10A cells.Signi?cantly Reduced Skin Carcinogenesis in Casp3(–/–)Mice after Two-Stage Chemical Carcinogenesis
We further conducted chemically induced carcinogenesis exper-iments in WT and caspase-3de?cient (Casp3[à/à])C57BL/6mice.We used a long established 7,12-Dimethylbenz(a)anthra-cene (DMBA)+12-O-tetradecanoyphobol-13-acetate
(TPA)
A D F
G
E
B
C
Figure 5.A Facilitative Role for Caspase-3in Radiation-Induced Oncogenic Transformation of MCF10A Cells
(A)Soft agar colony formation in MCF10A cells with high and low caspase-3reporter activities.Irradiated (0.5Gy of 600MeV 56Fe ions)cells were sorted for high and low caspase-3reporter expression by use of an FACS sorter.They were then cultured for 14days and seeded in soft agar plates.(B)The effects of shCASP3gene expression on soft agar colony formation in irradiated MCF10A cells.
(C)The effects of dominant-negative caspase-3(CASP3DN )expression on soft agar colony formation ability of irradiated MCF10A cells.
Error bars in (A)–(C)represent SEM.All values are derived from the average of three independent experiments.Student’s t test was used to calculate the p values in (A)–(C).
(D)A xenograft tumor in nude mice after 5weeks inoculation of irradiated MCF10A cells (indicated by arrow in red).
(E)H&E staining of tissues derived from a xenograft tumor formed from irradiated MCF10A cells in nude mice.The right panel shows an ampli?ed image of part of the left panel.
(F)Tumorigenic abilities of different MCF10A cells.Only the WT MCF10A cells irradiated with 0.5Gy can form tumors in nude mice.For each group of tumor cells,ten mice were used.
(G)Tumor growth kinetics from each of the ten mice injected with the WT MCF10A cells irradiated with 0.5Gy of 56Fe ions.There were six out of ten mice that showed tumor growth.See also Figure S5.
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two-stage carcinogenesis protocol following published proce-dures (Abel et al.,2009).In our induction regimen,an initial application of DMBA was followed by 20weeks of TPA adminis-tration.When the skins from C57/BL6mice that were treated with DMBA+TPA were analyzed for caspase-3activation,we observed a gradual increase in caspase-3activation that peaked at around 3weeks post treatment and attenuated afterward (Fig-ures 6A and 6B).In WT mice,DMBA+TPA induced numerous tu-mors,as expected.Tumor incidence was signi?cantly earlier and higher in WT mice than in Casp3(à/à)mice (Figures 6C and 6D).In addition,in Casp3-knockout (Casp3à/à)mice,the numbers of chemically induced tumors were signi?cantly reduced (Fig-ure 6E).Furthermore,the aggregate tumor sizes in WT mice were signi?cantly larger than those in the Casp3(à/à)mice (Fig-ure 6F).Figure S5D shows typical H&E staining of two tumor sec-tions from WT and Casp3(à/à)mice,respectively.
A careful analysis further reveals that there were clear sex dif-ferences in DMBA+TPA tumor induction in WT and Casp3KO (knockout or à/à)mice (Figures S6A–S6C).Female mice ap-peared to be more susceptible to DMBA+TPA induced tumors.In both sexes,caspase-3knockout caused profound reductions in tumorigenesis.However,female mice showed earlier and higher tumor incidence (Figure S6A),higher numbers of tumors per mouse (Figure S6B),and bigger tumor sizes (Figure S6C)in WT as well as Casp3KO mice.
We further compared the role of caspase-7with that of cas-pase-3in DMBA+TPA induced carcinogenesis.Our data sug-gest that Casp7knockout also led to signi?cantly
attenuated
A B
D E
F
C
Figure 6.A Facilitative Role for Caspase-3in Two-Stage Chemically Induced Skin Carcinogenesis in WT and Casp3(–/–)C57BL/6Mice
(A)Activated caspase-3in DMBA+TPA treated pre-malignant mouse skin.Scale bars,50m m.
(B)Quantitative measurement of caspase-3activation in DMBA+TPA treated C57BL/6mouse skin.Error bars show SD (n =3).
(C)Photographs representative of skin tumor formation in WT and Casp3(à/à)C57BL/6mice at 20weeks after the initiation of two-stage chemical treatment.(D)Tumor incidence in DMBA+TPA treated WT (n =23)and Casp3(à/à)(n =26)mice;p <0.001and log rank test.
(E)Average number of tumors per mouse in DMBA+TPA treated WT and Casp3(à/à)mice at 20weeks after initiation of chemical treatment.The difference between the two groups is statistically signi?cant (p <0.001and two-sided ANOVA test).
(F)Average tumor volume per mouse during the course of two-stage chemical induction.For each mouse,tumor burden represents the aggregate of all tumors in the mouse.The difference between the two groups is statistically signi?cant (p <0.001and two-sided ANOVA test).Error bars in (E)and (F)represent SEM.See also Figures S5and S6.
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carcinogenesis(Figures S6D–S6F).However,compared to cas-pase-3,knocking out caspase-7had a more moderate effect in terms of attenuating DMBA+TPA induced carcinogenesis,a result consistent with results from radiation-induced DNA dam-age experiments obtained with Casp7à/àmouse embryonic ?broblasts(Figures S2B and4E).
Endonuclease G as a Major Downstream Effector of Caspase-3-Mediated DNA Strand Breaks
We next attempted to determine the factors downstream of cas-pase-3that are responsible for generating DNA damage in cells with non-lethal activation of caspases3.We focused our atten-tion on Endonuclease G(EndoG),because it normally resides within the mitochondria and can migrate into the nucleus to frag-ment nuclear DNA in the event of caspase-3activation(Li et al., 2001;Parrish et al.,2001).
We?rst examined EndoG distribution through immuno?uores-cence staining.Our data indicated that most of the non-irradiated cells exhibited faint staining in the cytoplasmic region,with a small fraction showing nuclear staining.Cytoplasmic EndoG were localized mostly in the perinuclear regions that correlated with mi-tochrondria staining(Figure7A,top panels),consistent with pub-lished literature.In irradiated cells,there is a signi?cant increase in the fraction of cells with nuclear EndoG staining(Figure7A,lower panels),consistent with EndoG movement from the mitochondria into the nucleus.Caspase-3activity appears to be a major regu-lator of EndoG’s cytoplasmic to nuclear movement as attenuation of Casp3through either Casp3DN or shCasp3expression in irra-diated MCF10A cells signi?cantly reduced the fraction of cells with nuclear EndoG staining(Figure7B).Additional data using MEF cells with Casp3gene knockout further con?rmed the role of caspase-3in mediating radiation-induced nuclear migration of EndoG(Figures S7A and S7B).Importantly,when DMBA+TPA induced skin tumors were analyzed;tumor tissues showed signif-icantly more nuclear EndoG staining than adjacent normal skin tis-sue.In comparison,nuclear migration was signi?cantly reduced in tumors induced in Casp3à/àmice(Figures S7C and S7D), consistent with the hypothesis that EndoG nuclear migration was involved in DMBA+TPA induced carcinogenesis.
We also carried western blot analysis of EndoG location in MCF10A cell before and after irradiation(Figure7C).Our results indicate that radiation-induced a clear migration of EndoG to the cytoplasm as well as the nucleus.However,downregulation of caspase-3activity through Casp3DN or shCasp3expression signi?cantly reduced the migration.
In further analysis,we show that irradiated cells with nuclear EndoG staining contained signi?cantly higher proportion of cells with g H2AX foci($45%)when compared with those without nu-clear EndoG staining($5%,Figures7D and7E)at10days after irradiation,consistent with an important role for EndoG in inducing persistent DNA strand breaks.
In additional experiments,we examined whether EndoG was required for radiation-induced persistent genetic instability by use of MCF10A cells stably transduced with ENDOG targeted shRNAs(Figure S7E).Our results indicated that attenuation of EndoG expression signi?cantly lowered the fraction of cells with persistent g H2AX foci among irradiated MCF10A cells(Fig-ure7F),thereby establishing EndoG as the key downstream effector of caspase-3in causing DNA https://www.sodocs.net/doc/cf17399411.html,et assay showed reduced DNA strand breaks in irradiated MCF10A cells with ENDOG knockdown(Figure7G).These results were further supported by chromosome aberration analyses of MCF10A cells with shRNA knockdown(Figure7H;Table S2).Finally,we show that ENDOG knockdown also reduced the number of radiation-induced soft agar colonies from the MCF10A cells(Figure7I). The results of downregulating EndoG were demonstrated with three independent shRNAs against ENDOG(Figures S7E–S7G),suggesting that they were not likely from off-target effects.
DISCUSSION
Apoptosis and factors involved in the apoptotic machinery are generally considered tumor suppressive(Hanahan and Wein-berg,2011).However,the absence of mutations in the apoptosis inducing factors(Casp3,Casp9,or APAF)in patient-derived tu-mor samples suggests that these factors are not major obstacles for carcinogenesis in mammalian cells.The most striking aspect of the present study is that our results demonstrate that cas-pase-3can play an active role in promoting genetic instability and carcinogenesis.
How do we reconcile the fact that most previous studies support the notion of apoptosis-inducing factors being tumor suppressive with our?nding that caspase-3activation promotes carcinogenesis?To really understand these con?icting results, we need to reexamine the fundamental premise of the apoptosis-tumor suppressive hypothesis.At?rst glance it is a very straightforward scenario:cells are exposed to stress and activate the apoptosis program,they die,and get scavenged. The whole process should be anti-oncogenic because there is no in?ammation and no-leftover DNA damage.While this sce-nario is certainly true for cells that do die,an implied,but critical, assumption is that all cells that initiate the apoptotic process will die.While it is a reasonable assumption,it has not been care-fully examined except in C.elegans.It was shown that some C.elegans cells with caspase activation can actually survive,if they are not engulfed by surrounding cells(Hoeppner et al., 2001;Reddien et al.,2001).In addition,previous studies have unveiled non-apoptotic roles of caspase-3and its downstream caspase-dependent DNase(CAD)in muscle differentiation(Fer-nando et al.,2002;Larsen et al.,2010).In the present study,we show clear evidence that many irradiated mammalian cells with caspase-3activation can survive.It is in these surviving cells with caspase-3activation that one sees signi?cantly elevated levels of genetic instability and oncogenic transformation.
Our results are consistent with a recent report that indi-cates treatment of glioma or MEF cells with TRAIL or FasL,an apoptosis-inducing agent,causes increased DNA damage and mutagenesis that is caspase-8-dependent(Lovric and Hawkins, 2010).It is also consistent with another study,which shows that prolonged mitotic arrest triggers partial caspase activation that causes increased DNA damage(Orth et al.,2012).
In conclusion,in this study we?nd that many cells can survive caspase-3activation after exposure to ionizing radiation.We further reveal a surprising and unconventional function for cas-pase-3in mammalian cells exposed to radiation:causing and sus-taining DNA damage and facilitating oncogenic transformation. Molecular Cell58,284–296,April16,2015a2015Elsevier Inc.293
EXPERIMENTAL PROCEDURES
Cell Lines and Tissue Culture
Early passage,immortalized,non-transformed human breast epithelial cell line,MCF10A,was a kind gift from Dr.Hatsumi Nagasawa of Colorado State University(Fort Collins,CO).MCF10A growth medium was composed of Dul-becco’s modi?ed Eagle’s medium(DMEM)/F12(Sigma)supplemented with 5%donor horse serum(Sigma),20nanogram per milliliter(ng/ml)epidermal growth factor(EGF;R&D Systems),0.5m g/ml hydrocortisone(Sigma), 100ng/ml cholera toxin(Sigma),10m g/ml insulin(Invitrogen),and100units/ ml penicillin and100m g/ml streptomycin.For g H2AX foci assays,the cells were cultured in growth medium with only2%horse serum and without
EGF.
A B
C
D E
F G H I
Figure7.A Signi?cant Role for EndoG as a Downstream Factor of Caspase-3in Mediating Radiation-Induced DNA Damage and Transformation
(A)Immuno?uorescence co-staining of EndoG(green)and mitochondria(red)in control(top panels)and irradiated(lower panels)MCF10A cells.Insets,DAPI staining of the same slides.
(B)Effect of caspase-3inhibition(through CASP3DN or shCasp3expression)on radiation-induced EndoG migration from cytoplasmic to nuclear locations.
(C)Western blot analysis of EndoG in the mitochondria,cytoplasmic,and nuclear fractions of MCF10A cells with or without Casp3DN or shCasp3expression. Mito marker,TATA binding protein(TBP),and b-actin were used as mitochondria,nuclear,and cytoplasmic loading controls,respectively.
(D)Immuno?uorescence co-staining of EndoG(green)and g H2AX foci(red)in irradiated MCF10A cells.Insets,DAPI staining of the same slides.
(E)Fraction of cells with g H2AX foci among cells with EndoG staining in the cytoplasm only or those in both the cytoplasm and nucleus.
(F)Effect of attenuating EndoG expression on radiation-induced g H2AX foci in MCF10A cells.
(G)Effects of attenuating EndoG expression on radiation-induced comet tail DNA amount(an estimate of DNA strand breaks)in MCF10A cells.
(H)Effects of attenuating EndoG expression on the fraction of cells with radiation-induced chromosome aberrations in MCF10A cells.
(I)Effects of attenuating EndoG expression on the soft agar colony forming abilities of irradiated MCF10A cells.
(F–I)shEndoG represents shRNA2from Figure S7F.
Error bars in(B)and(E)–(I)represent SEM(n=3for each data point).For g H2AX foci,at least200cells were counted for each measurement.For comet assay,a minimum of50cells were randomly chosen and measured automatically by Image J software(NIH)for DNA distribution.For chromosome aberration analysis,a minimum of150cells were counted without knowledge of cell identities.Student’s t test was used to calculate the p values.See also Figure S7.
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Exposure to Higher Energy56Fe Ions
To conduct irradiation with56Fe ions,cells to be irradiated or sham-irradiated were shipped by FedEx to the National Aeronautics and Space Administration (NASA)-sponsored Space Radiation Laboratory at Brookhaven National Lab-oratory(BNL;Brookhaven,Long Island,NY)in sealed T-25?asks.The iron beam energy used was600MeV/m.The dose rate for exposure was0.5Gy/ min.After irradiation,the cells were immediately shipped back to our labora-tory in Durham,NC for further analysis.
g H2AX Foci Assay
To detect the radiation-induced DSBs,g H2AX foci in the irradiated cells were examined through immuno?uorescence by use of an established protocol (Paull et al.,2000;Rogakou et al.,1998).
Alkaline Comet Assay
We also used alkaline comet assay(Olive et al.,1990;Ostling and Johanson, 1984;Singh et al.,1988)to detect DSBs.To do this,we used a commercial kit following the manufacturer’s(Trevigen)instructions.
Chromosome Aberration Analysis
We carried out chromosome aberration analysis in cultured cells and in bone marrow cells irradiated in mice.We also analyzed chromosome translocations analysis by use of?uorescence in situ hybridization(FISH)analysis in bone marrow cells from sham and irradiated mice.
Two-Stage Carcinogenesis
An established protocol(Abel et al.,2009)was used to induce skin carcinogen-esis in mice by use of combined DMBA+TPA administration onto shaved mouse skin.An initial DMBA treatment and periodic(23weekly)TPA treat-ments were carried.At the end of20weeks,the number of tumor per mice and tumor sizes were enumerated and quanti?ed,respectively.All animal ex-periments described in this study has been approved by the Duke University Institutional Animal Care and Use Committee.
Statistical Analysis
Speci?c statistical methods are mostly described in the?gure legends.Where it is not stated,two-tailed Student’s t test was used to compare differences be-tween two groups.In other instances,ANOVA or log rank tests were also used and described in the?gure legends.
Please also see Supplemental Experimental Procedures for additional de-tails on various experimental methods used in this study.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures, seven?gures,and three tables and can be found with this article online at https://www.sodocs.net/doc/cf17399411.html,/10.1016/j.molcel.2015.03.003.
AUTHOR CONTRIBUTIONS
X.L.,Y.H.,and C.-Y.L.conceived of the study and designed the experiments.X.L. carried out most of the experiments.Y.H.carried out some of the initial studies and key initial carcinogenesis experiments.F.L.and Q.H.carried out some of the soft agar studies.T.A.K.carried out some of the chromosome aberrations analysis.R.P.H.provided intellectual input in designing some of the experiments and helped in writing the manuscript.X.L.and C.-Y.L.wrote the manuscript.
ACKNOWLEDGMENTS
This study was supported in part by grants CA131408,CA136748,CA155270, and ES024015from the NIH(to C.-Y.L.)and grant NNX12AB88G(to C.-Y.L.) from the NASA Space Radiation Biology Research Program,the Duke Skin Disease Research Core Center grant(AR066527)from the NIH to R.P.H., and grants30428015and30325043from the National Science Foundation of China and grant2004CB518804from the Ministry of Science of China ‘‘973project’’to Q.H.
We thank Dr.Sally Kornbluth(Duke University)for reading our manuscript and providing insightful suggestions.We also thank Dr.Richard A.Flavell (Yale University)for depositing the CASP3à/àmouse to Jackson Laboratory for research use;Drs.Guy Salvesen(Sanford Burnham Insti-tute),David M.Spencer(Baylor College of Medicine),Chris Counter (Duke University),and Titia de Lange(Rockefeller University)for sharing their CASPDN,iCasp3,p53DN,and53BP1-mCherry plasmids,respec-tively,through Addgene;and Drs.Adam Rusek and Peter Guida for their help in carrying out56Fe ion irradiation of our cells at the Brookhaven National Laboratory.
Received:October1,2014
Revised:December24,2014
Accepted:February18,2015
Published:April9,2015
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caspase-3
(一)caspase家族 Caspases是近年来发现的一组存在于胞质溶胶中的结构上相关的半胱氨酸蛋白酶，它们的一个重要共同点是特异地断开天冬氨酸残基后的肽键。Caspase一词是从Cysteine aspartic acid specific protease 的字头缩写衍生而来，就反映了这个特征，而这种高度的特异性，在蛋白酶中是很少见的。由于这种特异性，使caspase能够高度选择性地切割某些蛋白质，这种切割只发生在少数（通常只有1个）位点上，主要是在结构域间的位点上，切割的结果或是活化某种蛋白，或使某种蛋白失活，但从不完全降解一种蛋白质。 Caspase的研究源于线虫（C. elegans）细胞程序化死亡的研究。线虫在发育过程中，有131个细胞将进入程序化死亡；研究发现有11个基因与PCD 有关，其中ced3和ced4基因是决定细胞凋亡所必需的，ced9基因抑制PCD。线虫细胞程序化死亡的研究促进了其他动物特别是哺乳类动物中细胞凋亡的研究。人们发现哺乳类细胞中存在着Ced3的同源物ICE（interleukin-1b converting enzyme），它催化白介素-1b的活化，即从其前体上将IL-1b 切割下来。在大鼠成纤维细胞中过量表达ICE和Ced3都会引起细胞凋亡，表明了ICE和Ced3在结构和功能上的相似性；然而敲除ICE基因的小鼠其表现型正常，并未发现细胞凋亡发生明显改变。进一步的研究发现，另一个ICE成员，后来被称为apopain，CPP32或Yama的半胱氨酸蛋白酶，催化poly（ADP-ribose）Polymerase（PARP），即聚（ADP-核糖）聚合酶的裂解，结果导致细胞的凋亡，因而认为apopain执行着与线虫中的ced3相同的功能。Apopain被称为是“死亡酶”，而PARP被认为是“死亡底物”。 Apopain/CPP32/Yama是在1995年由两个实验室分别同时报导，时间上只有两周之差。Ced4的哺乳类同源物则迟迟未能发现，直到1997年，才被证明是Apaf-1（即一种细胞凋亡蛋白酶活化因子apoptosis protease activating factor）。而Ced 9的哺乳类对应物则较早地被证明是BCL-2，这一问题将在以后的部分述及。 现已确定至少存在11种caspase： 这些caspases中，caspase 1和caspase 11，以及可能还有caspase 4被认为不直接参与凋亡信号的转导，它们主要参与白介素前体的活化；而caspase 2，caspase 8，caspase 9和caspase 10参与细胞凋亡的起始；参与细胞凋亡执行的则是caspase 3，caspase 6和caspase 7，其中caspase 3和7具有相近的底物和抑制剂特异性，它们降解PARP，DFF-45（DNA fragmentation factor-45），导致DNA修复的抑制并启动DNA的降解。而
酸性蛋白酶的作用机理
酸性蛋白酶与碱性蛋白酶生产工艺的不同之处？ 酸性蛋白酶是一种在酸性环境下（pH 2.5-4.0）催化蛋白酶水解的酶制剂，适用于酸性介质中水解动植物蛋白质。可用于毛皮软化，酒精发酵，啤酒、果酒澄清，动植物蛋白质水解营养液，羊毛染色，废胶片回收，饲料添加剂等等。本品在酸性条件下有利于皮纤维松散，且软化液可连续使用，是当前理想的毛皮软化酶制剂；在酒精发酵中，添加酸性蛋白酶，能有效水解原料中的蛋白质，破坏原料颗粒粒间细胞壁的结构，有利于糖化酶的作用，使原料中可利用碳源增加，从而可提高原料出酒率；另一方面，蛋白质的水解提高了醪液中α-氨基态氮的含量，促进酵母菌的生长与繁殖，提高发酵速度，从而缩短发酵周期和提高发酵设备的生产能力。 碱性蛋白酶碱性蛋白酶是在碱性条件下水解蛋白质肽键的酶类,是一类非常重要的工业用酶,最早发现于猪胰脏。碱性蛋白酶广泛存在于动、植物及微生物中。微生物蛋白酶均为胞外酶,不仅具有动植物蛋白酶所具有的全部特性,还有下游技术处理相对简单、价格低廉、来源广、菌体易于培养、产量高、高产菌株选育简单、快速、易于实现工业化生产等诸多优点。1945年瑞士M等在地衣芽孢杆菌中发现了微生物碱性蛋白酶。 碱性蛋白酶是由细菌原生质体诱变选育出的地衣芽孢杆菌2709，经深层发酵、提取及精制而成的一种蛋白水解酶，其主要酶成分为地衣芽孢杆菌蛋白酶，是一种丝氨酸型的内切蛋白酶，它能水解蛋白质分子肽链生成多肽或氨基酸，具有较强的分解蛋白质的能力，广泛应用
于食品、医疗、酿造、洗涤、丝绸、制革等行业。 1、碱性蛋白酶是一种无毒、无副作用的蛋白质，属于丝氨酸型内切蛋白酶，应用在食品行业可水解蛋白质分子肽链生成多肽或氨基酸，形成具有独特风味的蛋白质水解液。 2、碱性蛋白酶成功应用于洗涤剂用酶工业，可添加在普通洗衣粉、浓缩洗衣粉和液体洗涤剂当中，既可用于家庭洗衣，也可用于工业洗衣，可以有效的去除血渍、蛋类、乳制品、或肉汁、菜汁等蛋白类的污渍，另外也可作为医用试剂酶清洗生化仪器等。 3、在生物技术领域，碱性蛋白酶可作为工具酶用于核酸纯化过程中的蛋白质（包括核酸酶类）去除，而对DNA无降解作用，避免对DNA 完整性的破坏。 酸性蛋白酶如何灭活第一种方法几乎所有酶都适用，就是加热。第二种，既然是酸性酶，加入强碱应该也是可以的。 酸性蛋白酶产生菌的筛选方法？酸性蛋白酶是一种能在酸性环境下水解蛋白质的酶类，其最适作用pH值为2.5-5.0。由于酸性蛋白酶具有较好的耐酸性，因此被广泛地应用于食品、医药、轻工、皮革工艺以及饲料加工工业中。目前用于工业化生产的酸性蛋白酶大多为霉菌酸性蛋白酶，此类酶的最适作用pH值为3.0左右，当pH值升高时，酸性蛋白酶的酶活会明显降低，且此类酶不耐热，当温度达到50℃以上时很不稳定，从而限制了酸性蛋白酶的应用范围。因此，本研究以开发耐温偏酸性蛋白酶为目标，进行了以下几方面的研究：(1)偏酸性蛋白酶产生菌的分离筛选。(2)偏酸性蛋白酶粗酶酶学性质的
caspase家族及在细胞凋亡中的作用
caspase家族及在细胞凋亡中的作用 admin 注:本文仅是基础知识，目前这方面进展十分快，需要更新更多的知识。如caspase家族目前发现至少14种。这里仅介绍几种常见的成员。目的是让大家了解到caspase家族的概况，以及在细胞凋亡中的作用。 一caspase家族蛋白酶的组成 未活化的caspase家族蛋白酶是以酶原形式存在的，酶原的氨基端有一段被称为“原结构域”(pro-domain)的序列。酶原活化时不但要将原结构域切除，并且要将剩余部分剪切成一大一小两个亚基，分别称为P20和P10，活性酶就是由这两种亚基以(P20/P10) 2 的形式组成的。这种活化反应也是Asp特异的，剪切发生在酶原中保守序列的Asp与其后的氨基酸残基之间，一般是先切下羧基端的小亚基，然后再从大亚基的氨基端切去原结构域。这种剪切可以是酶原及中间活性酶自我催化，也可以是其它ICE家族蛋白酶的作用，还有其它酶类如颗粒酶B 参与。 表5. caspase家族蛋白酶的识别序列及作用底物 蛋白酶名称别名识别序 列 底物 caspase 1ICE YVAD pro-IL-1 , pro-caspase 3, pro-caspase 4 caspase 4TX, ICH-2, ICErel-II pro-caspase 1 caspase 5ICErel-III, TY mICH3 mICH4 caspase 2ICH-1PARP caspase 9ICE-LAP6, Mch6PARP
caspase 3CPP32, Yama, apopain DEVD PARP, DNA-PK, SRE/BP, rho-GDI, KCθ caspase 6Mch2VEID lamin A caspase 7Mch3, ICE-LAP3, CMH-1 DEVD PARP, pro-caspase 6 caspase 8MACH, FLICE, Mch5DEVD pro-caspase3,4,7,9,10 caspase 10Mch4YVAD caspase 11ICH3, FLICE2 CED-3 已命名的caspase家族成员均已克隆成功，它们不但在氨基酸序列上具有同源性，而且在空间结构上也很相似。目前已经获得了caspase-1(ICE)和caspase-3(CPP32)的X线结晶图像，结果显示它们具有相似的空间结构，在P20的C端和P10的N端有200多个氨基酸残基的序列尤为保守，在空间结构上组成相似的β折叠中心和相邻的α螺旋，保守的、对蛋白酶活性有特殊意义的氨基酸残基均位于这一段，并形成特定的结构。不同源的序列主要存在于P20的N 端和P20与P10交界处，这两个部位的氨基酸残基在蛋白酶活化过程中一般被全部或部分切除。 所有的成员都保守性地包含有与底物P 1 Asp作用的氨基酸残基，如在ICE中，它们是催化中心的Cys285，与酶/抑制剂复合物的巯基半缩醛以氢键结合的His237，以及可以稳定反应中间物氧 阴离子的Gly238。另外，Arg179、Arg341、Gln383和Ser347形成容纳P 1 Asp的“口袋”，Ser339靠近 Cys285的巯基，以氢键结合P 1位的酰胺。与P 2 ~P 4 作用的氨基酸残基则变异比较大，这可能是各 个成员识别不同底物的特异性之所在。在活性Cys周围的氨基酸序列也很保守，一般都有Gln-Ala-Cys-Arg-Gly(QACRG)五肽序列，在caspase-8、caspase-10中为QACQG，在caspase-9中是QACGG，这三个成员都有一个氨基酸残基的变异，但这种变异不影响蛋白酶的剪切活性和特异性。
caspase资料
caspase家族蛋白酶的组成 已命名的caspase家族成员均已克隆成功，它们不但在氨基酸序列上具有同源性，而且在空间结构上也很相似。目前已经获得了caspase-1(ICE)和caspase-3(CPP32)的X线结晶图像，结果显示它们具有相似的空间结构，在P20的C端和P10的N端有200多个氨基酸残基的序列尤为保守，在空间结构上组成相似的β折叠中心和相邻的α螺旋，保守的、对蛋白酶活性有特殊意义的氨基酸残基均位于这一段，并形成特定的结构。不同源的序列主要存在于P20的N端和P20与P10交界处，这两个部位的氨基酸残基在蛋白酶活化过程中一般被全部或部分切除。 所有的成员都保守性地包含有与底物P1Asp作用的氨基酸残基，如在ICE中，它们是催化中心的Cys285，与酶/抑制剂复合物的巯基半缩醛以氢键结合的His237，以及可以稳定反应中间物氧阴离子的Gly238。另外，Arg179、Arg341、Gln383和Ser347形成容纳P1Asp的“口袋”，Ser339靠近Cys285的巯基，以氢键结合P1位的酰
胺。与P2~P4作用的氨基酸残基则变异比较大，这可能是各个成员识别不同底物的特异性之所在。在活性Cys周围的氨基酸序列也很保守，一般都有Gln-Ala-Cys-Arg-Gly(QACRG)五肽序列，在caspase-8、caspase-10中为QACQG，在caspase-9中是QACGG，这三个成员都有一个氨基酸残基的变异，但这种变异不影响蛋白酶的剪切活性和特异性。 未活化的caspase家族蛋白酶是以酶原形式存在的，酶原的氨基端有一段被称为“原结构域”(pro-domain)的序列。酶原活化时不但要将原结构域切除，并且要将剩余部分剪切成一大一小两个亚基，分别称为P20和P10，活性酶就是由这两种亚基以(P20/P10)2的形式组成的。这种活化反应也是Asp特异的，剪切发生在酶原中保守序列的Asp与其后的氨基酸残基之间，一般是先切下羧基端的小亚基，然后再从大亚基的氨基端切去原结构域。这种剪切可以是酶原及中间活性酶自我催化，也可以是其它ICE家族蛋白酶的作用，还有其它酶类如颗粒酶B参与。二caspase家族蛋白酶的结构与功能 按照结构同源性的大小，可以将caspase蛋白酶分为三个组，分别以caspase-1、caspase-2和caspase-3为代表。其中最重要的是caspase-1、caspase-3 和caspase-8。
蛋白酶的种类
蛋白酶的论述 摘要：蛋白酶（英语：Protease）是生物体内的一类酵素（酶），它们能够分解蛋白质。分解方法是打断那些将氨基酸连结成多肽链的肽键。抑制蛋白酶活性的小分子化合物被称蛋白酶抑制剂。许多病毒蛋白酶的抑制剂是很有效的抗病毒药。 1．木瓜蛋白酶 1.1木瓜蛋白酶简介 木瓜蛋白酶，是一种蛋白水解酶，可将抗体分子水解为3个片段。是番木瓜中含有的一种低特异性蛋白水解酶，活性中心含半胱氨酸，属巯基蛋白酶，应用于啤酒及食品工业。 1.2木瓜蛋白酶的特点 木瓜蛋白酶（Papain）简称木瓜酶，又称为木瓜酵素。是利用未成熟的番木瓜（Carica papaya）果实中的乳汁，采用现代生物工程技术提炼而成的纯天然生物酶制品。它是一种含巯基(-SH)肽链内切酶，具有蛋白酶和酯酶的活性，有较广泛的特异性，对动植物蛋白、多肽、酯、酰胺等有较强的水解能力，同时，还具有合成功能，能把蛋白水解物合成为类蛋白质。溶于水和甘油，水溶液无色或淡黄色，有时呈乳白色；几乎不溶于乙醇、氯仿和乙醚等有机溶剂。最适合PH值6～7(一般3～9.5皆可)，在中性或偏酸性时亦有作用，等电点（pI)为8.75；最适合温度55～65℃(一般10～85℃皆可)，耐热性强，在90℃时也不会完全失活；受氧化剂抑制，还原性物质激活。木瓜蛋白酶由212个氨基酸残基组成，当用氨基肽酶从N末端水解掉分子中的2/3肽链后，剩下的1/3肽链仍保持99%的活性，说明木瓜蛋白酶的生物活性集中表现在C末端的少数氨基酸残基及其所构成的空间结构区域。 木瓜蛋白酶papain属巯基蛋白酶，具有较宽的底物特异性，作用于蛋白质中L-精氨酸、L-赖氨酸、甘氨酸和L-瓜氨酸残基羧基参与形成的肽键。此酶属内肽酶，能切开全蛋蛋白质分子内部肽链—CO—NH—生成分子量较小的多肽类。存在于木瓜胚乳中的蛋白酶。EC3．4．22．2。作为植物来源的蛋白酶来说，此酶研究进展的最快。此酶主要是以内肽酶的形态起作用。活性的产生，而半胱氨酸残基是不可缺少的，所以是硫基蛋白酶的一种，底物的特异性不太严格，分子量为23400，氨基酸残基数212。 木瓜蛋白酶是一种在酸性、中性、碱性环境下均能分解蛋白质的蛋白酶。它的外观为白色至浅黄色的粉末，微有吸湿性。 酪蛋白被木瓜蛋白酶降解生成的酪氨酸在紫外光区 275nm 处有吸收峰。1.3木瓜蛋白酶物理化学性质 本品为乳白色至微黄色粉末，具有木瓜特有的气味,稍具有吸湿性。水解蛋白质能力强,但几乎不能分解蛋白胨，易溶于水,甘油,不溶于一般的有机溶剂,耐热性强。由木瓜制得的商品酶制剂中，含有如下三种酶：(1)木瓜蛋白酶，分
猪Caspase_3基因的克隆及序列分析
收稿日期:20060726 基金项目:广西大学科学技术研究基金重点资助项目(2003ZD 01);广西科技攻关项目(桂科攻0480001) 作者简介:李军(1971),男,广东梅州人,助理研究员,博士研究生,研究方向为动物传染病防治与分子病毒学。*为通讯作者。 猪Caspase -3基因的克隆及序列分析 李 军, 刘 兵, 曾 兰, 罗廷荣 * (广西大学动物科学技术学院, 南宁 530005) 摘要:天冬氨酸特异性半胱氨酸蛋白酶(Caspase)是与细胞凋亡有关的一类蛋白酶,其中Caspase-3是细胞凋亡的最终执行者。为了进一步研究Caspase-3的生物学功能,从P K-15细胞中提取总RN A ,用RT -PCR 方法扩增出猪Caspase-3基因,序列分析表明克隆的Caspase-3基因与GenBank 上登录的猪Caspase-3基因核苷酸与推导的氨基酸序列同源性分别为98.9%和98.6%,与人和鼠的Ca spase -3基因核苷酸、氨基酸序列同源性分别为88.6%、83.5%和87.8%、86.7%,而且猪、人、鼠的Caspase-3上的催化和剪切位点都极为保守。 关键词:Caspase-3;克隆;序列分析中图分类号:S 813.1 文献标识码:A 文章编号:1002—8161(2006)05-0584-05 Cloning and sequence analysis of Caspase -3gene of pig LI Jun ,LIU Bing ,ZENG Lan ,LU O Ting -r ong * (A nimal Science and T echnology College ,Guang x i Univers ity ,N anning 530005,China ) Abstract :Caspase is a kind of pr ot eases which is r elated to apopto sis ,and Caspase -3is the ultimate ex ecute in apoptosis.In or der to st udy t he bio log ical functio n o f Caspase-3,t he Caspase-3gene of pig wa s amplified by RT -PCR w it h the ex tr actio n o f RN A fr om P K -15cells.Aft er clo ning and sequencing ,the results show ed that t he nu-cleo tide a nd the deduced amino acid sequences of clo ned Caspase -3sha red 98.9%and 98.6%homo log y w ith those of Caspase -3published in GenBank .A s co mpar ed with that of human a nd mouse ,the Caspase -3gene homo lo gy of the nucleotide and the deduced amino acid sequences w er e 88.6%,83.5%and 87.8%,86.7%,respect ively.T he activ e sites cor responding catalysis and cleav ag e o n Caspase-3amo ng the pig ,human and mo use w ere conser vative ver y much . Key words :Caspase -3;cloning ;sequence a nalysis 天冬氨酸特异性半胱氨酸蛋白酶(aspartate-specific cy steinyl pro teinase,Caspase)家族是执行 细胞凋亡的一类重要蛋白酶。Caspase 通常以单一的蛋白酶原前体形式存在,被激活后引起一系列酶级联效应,特异性地在特定的氨基酸序列中将肽链从天冬氨酸(Asp )之后切断,还可引起染色体DNA 的降解及细胞的解体[1,2]。在细胞凋亡信号传导的Caspase 级联反应中,Caspase-3是凋亡信号传导途径下游的一个关键效应因子,它可以被Fas /FasL 途径活化,也可经颗粒酶B 途径活化。研究表明,Caspase-3可以导致所有类型的细胞发生染色质浓缩和形成DNA 片段化[3,4]。此外,Caspase -3可以抑 制白细胞介素18的生物学活性[5],因此Caspase-3在细胞凋亡和炎症反应中起到关键的作用。 猪的许多病毒病都涉及到细胞凋亡[6～9],有的病毒既能促进细胞凋亡又能抑制细胞凋亡;有的只能诱导细胞凋亡;而有的只能抑制细胞凋亡。但是Caspase-3在其中的分子作用机制尚未完全清楚,本研究通过克隆猪的Caspase-3基因,以便进一步研究在病毒感染过程中Caspase -3对细胞凋亡的影响。 1 材料与方法 1.1 主要试剂 RNA 抽提试剂Tr izol Reag ent 、T aq DNA 聚合酶,购自上海生工生物工程技术服务
蛋白酶的种类
蛋白酶的种类 1．木瓜蛋白酶 木瓜蛋白酶，是一种蛋白水解酶，可将抗体分子水解为3个片段。是番木瓜中含有的一种低特异性蛋白水解酶，活性中心含半胱氨酸，属巯基蛋白酶，应用于啤酒及食品工业。 木瓜蛋白酶papain属巯基蛋白酶，具有较宽的底物特异性，作用于蛋白质中L-精氨酸、L-赖氨酸、甘氨酸和L-瓜氨酸残基羧基参与形成的肽键。此酶属内肽酶，能切开全蛋蛋白质分子内部肽链—CO—NH—生成分子量较小的多肽类。 木瓜蛋白酶是一种在酸性、中性、碱性环境下均能分解蛋白质的蛋白酶。它的外观为白色至浅黄色的粉末，微有吸湿性。 木瓜蛋白酶（Papain）简称木瓜酶，又称为木瓜酵素。是利用未成熟的番木瓜（Carica papaya）果实中的乳汁，采用现代生物工程技术提炼而成的纯天然生物酶制品。它是一种含疏基(-SH)肽链内切酶，具有蛋白酶和酯酶的活性，有较广泛的特异性，对动植物蛋白、多肽、酯、酰胺等有较强的水解能力，同时，还具有合成功能，能把蛋白水解物合成为类蛋白质。溶于水和甘油，水溶液无色或淡黄色，有时呈乳白色；几乎不溶于乙醇、氯仿和乙醚等有机溶剂。最适合PH值6～7(一般3～9.5皆可)，在中性或偏酸性时亦有作用，等电点（pI)为8.75；最适合温度55～65℃(一般10～85℃皆可)，耐热性强，在90℃时也不会完全失活；受氧化剂抑制，还原性物质激活。
2．胃蛋白酶 胃蛋白酶（英文名称：Pepsin）是一种消化性蛋白酶，由胃部中的胃粘膜主细胞所分泌，功能是将食物中的蛋白质分解为小的肽片段。胃蛋白酶原由胃底主细胞分泌，在pH1.5～5.0条件下，被活化成胃蛋白酶，将蛋白质分解为胨，而且一部分被分解为酪氨酸、苯丙氨酸等氨基酸。可分解蛋白质中苯丙氨酸或酪氨酸与其他氨基酸形成的肽键，产物为蛋白胨及少量的多肽和氨基酸，该酶的最适pH为2左右。 3．中性蛋白酶 中性蛋白酶是由枯草芽孢杆菌经发酵提取而得的，属于一种内切酶，可用于各种蛋白质水解处理。在一定温度、PH值下，本品能将大分子蛋白质水解为氨基酸等产物。可广泛应用于动植物蛋白的水解，制取生产高级调味品和食品营养强化剂的HAP和HVP，此外还可用于皮革脱毛、软化、羊毛丝绸脱胶等加工。 利用中性蛋白酶的酶促反应，可把动植物的大分子蛋白质水解成小分子肽或氨基酸，以利于蛋白质的有效吸收和利用，其水解液AN%高，水解度高，风味佳，已广泛用于生产高级调味品和食品营养强化剂，各种动物来源性抽提物生产功能性骨、肉提取物（骨素）、水产提取物、蛋白胨、肽等及研究开发一些高附加值的功能食品。
人半胱氨酸蛋白酶-3(caspase-3)试剂盒使用方法
人半胱氨酸蛋白酶-3（caspase-3）试剂盒使用方法本试剂盒仅供研究使用。 检测范围：96T 3 pmol/L -80 pmol/L 使用目的： 本试剂盒用于测定人血清、血浆及相关液体样本中半胱氨酸蛋白酶-3（caspase-3）含量。实验原理 本试剂盒应用双抗体夹心法测定标本中人半胱氨酸蛋白酶-3（caspase-3）水平。用纯化的人半胱氨酸蛋白酶-3（caspase-3）抗体包被微孔板，制成固相抗体，往包被单抗的微孔中 依次加入半胱氨酸蛋白酶-3（caspase-3），再与HRP 标记的半胱氨酸蛋白酶-3（caspase-3）抗体结合，形成抗体-抗原-酶标抗体复合物，经过彻底洗涤后加底物TMB 显色。TMB 在HRP 酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。颜色的深浅和样品中的半胱氨酸蛋白酶-3（caspase-3）呈正相关。用酶标仪在450nm 波长下测定吸光度（OD 值），通过标准曲线计算样品中人半胱氨酸蛋白酶-3（caspase-3）浓度。 试剂盒组成 1 30 倍浓缩洗涤液 20ml×1 瓶 7 终止液 6ml×1 瓶 2 酶标试剂 6ml×1 瓶 8 标准品（160 pmol/L） 0.5ml×1 瓶 3 酶标包被板 12 孔×8 条 9 标准品稀释液 1.5ml×1 瓶 4 样品稀释液 6ml×1 瓶 10 说明书 1 份 5 显色剂A 液 6ml×1 瓶 11 封板膜 2 张 6 显色剂B 液 6ml×1/瓶 12 密封袋 1 个 标本要求 1．标本采集后尽早进行提取，提取按相关文献进行，提取后应尽快进行实验。若不能 马上进行试验，可将标本放于-20℃保存，但应避免反复冻融 2．不能检测含NaN3 的样品，因NaN3 抑制辣根过氧化物酶的（HRP）活性。 操作步骤 1. 标准品的稀释：本试剂盒提供原倍标准品一支，用户可按照下列图表在小试管中进行稀释。 80 pmol/L 5 号标准品 150μl 的原倍标准品加入150μl 标准品稀释液 40 pmol/L 4 号标准品 150μl 的5 号标准品加入150μl 标准品稀释液 20 pmol/L 3 号标准品 150μl 的4 号标准品加入150μl 标准品稀释液 10 pmol/L 2 号标准品 150μl 的3 号标准品加入150μl 标准品稀释液 5 pmol/L 1 号标准品 150μl 的2 号标准品加入150μl 标准品稀释液 2. 加样：分别设空白孔（空白对照孔不加样品及酶标试剂，其余各步操作相同）、标准孔、待测样品孔。在酶标包被板上标准品准确加样50μl，待测样品孔中先加样品稀释液40μl，然后再加待测样品10μl（样品最终稀释度为5 倍）。加样将样品加于酶标板孔底部，尽 量不触及孔壁，轻轻晃动混匀。 3. 温育：用封板膜封板后置37℃温育30 分钟。 4. 配液：将30 倍浓缩洗涤液用蒸馏水30 倍稀释后备用 5. 洗涤：小心揭掉封板膜，弃去液体，甩干，每孔加满洗涤液，静置30 秒后弃去，如此重复5 次，拍干。
糜蛋白酶
药品名称：糜蛋白酶Chymotrypsin 药品分类：呼吸系统类→祛痰药物→促进痰液溶解的药物 药品别名：胰凝乳蛋白酶、α-糜蛋白酶、Avazyme、Chymar 药品剂型：注射用糜蛋白酶：1mg(800U)；5mg(4000U)。 药理作用：糜蛋白酶是由牛胰中分离制得的一种蛋白分解酶类药，作用与胰蛋白酶相似，能促进血凝块、脓性分泌物和坏死组织等的液化清除。本药具有肽链内切酶及脂酶的作用：可将蛋白质大分子的肽链切断，成为分子量较小的肽，或在蛋白分子肽链端上作用，使氨基酸分出；并可将某些脂类水解。通过此作用能使痰中纤维蛋白和粘蛋白等水解为多肽或氨基酸，使粘稠痰液液化，便于咳出，对脓性或非脓性痰都有效。此外，本药尚能松弛睫状韧带及溶解眼内某些组织的蛋白结构。糜蛋白酶还有促进抗生索、化疗药物向病灶渗透的作用。 药动学：本药和胰蛋白酶都是强力蛋白水解酶，仅水解部位有差异。蛇毒神经毒含碱性氨基酸，易被本药和胰蛋白酶分解为无毒蛋白质，从而阻断毒素进入血流产生中毒作用。本药对蝮亚科蛇伤疗效优于胰蛋白酶，两种酶制剂联合应用效果更佳。 适应症：1.用于眼科于术松弛睫状韧带、减轻创伤性虹膜睫状体炎；也可用于白内障摘除，使晶体易于移去。2.用于创伤或手术后伤口愈合、抗炎及防止局部水肿、积血、扭伤血肿、乳房手术后浮肿、中耳炎、鼻炎等。3.用于慢性支气管炎、支气管扩张或肺脓肿的治疗，可使脓性和非脓性痰液均可液化，易于咳出。4.毒蛇咬伤的处理。 禁忌症：1.20岁以下的患者，由于晶体囊膜玻璃体韧带相连牢固，眼球较小，巩膜弹性强可致玻璃体脱出，或玻璃体液不固定的创伤性白内障病人，因可导致玻璃体液丧失，故均禁用。 2.眼压高或伴有角膜变性的白内障患者，以及玻璃体有液化倾向者均禁用。 3.严重肝、肾疾病、凝血功能异常及正在应用抗凝药者禁用。 注意事项：1.本药肌内注射前需做过敏试验，并禁止静脉注射。2.如引起过敏反应，应立即停止使用，并用抗组胺类药物治疗。3.本药对视网膜有较强的毒性，由于可造成晶体损坏，应用时勿使药液透入玻璃体。4.本药遇血液迅速失活，因此在用药部位不得有末凝固血液。 5.本药在固体状态时比较稳定，但其溶液不稳定，室温放置9天可损失50%的活性，故应临用前配制。 6.对本药引起的青光眼症状，于术后滴用β-受体阻滞药(如噻吗洛尔)或口服碳酸酐酶抑制药(如乙酰唑胺)，可能会缓解。 7.由于超声雾化后糜蛋白酶效价下降明显，因此，糜蛋白酶超声雾化吸入时间宜控制在5min内。 不良反应：1.眼：眼科局部用药一般不会引起全身不良反应，但可引起短期眼压增高，导致眼痛、眼色素膜炎和角膜水肿，这种青光眼症状可持续1周后消退，还可导致角膜线状混浊、玻璃体疝、虹膜色素脱落、葡萄膜炎及创口开裂或延迟愈合等。2.血液系统：糜蛋白酶可造成凝血功能障碍。3.其他：(1)肌内注射偶可致过敏性休克。(2)糜蛋白酶可引起组胺释放，导致局部注射部位疼痛、肿胀。 用法用量：[该用法是参考最新药典提供，临床中具体药物用法用量请参考药物说明书] 成人常用量1.肌内注射：通常一次4000U，用前将糜蛋白酶以氯化钠注射液5ml溶解。2.
一 caspase家族蛋白酶的组成
一caspase家族蛋白酶的组成 未活化的caspase家族蛋白酶是以酶原形式存在的，酶原的氨基端有一段被称为“原结构域”(pro-domain)的序列。酶原活化时不但要将原结构域切除，并且要将剩余部分剪切成一大一小两个亚基，分别称为P20和P10，活性酶就是由这两种亚基以(P20/P10)2的形式组成的。这种活化反应也是Asp特异的，剪切发生在酶原中保守序列的Asp与其后的氨基酸残基之间，一般是先切下羧基端的小亚基，然后再从大亚基的氨基端切去原结构域。这种剪切可以是酶原及中间活性酶自我催化，也可以是其它ICE家族蛋白酶的作用，还有其它酶类如颗粒酶B参与。 表5. caspase家族蛋白酶的识别序列及作用底物
已命名的caspase家族成员均已克隆成功，它们不但在氨基酸序列上具有同源性，而且在空间结构上也很相似。目前已经获得了caspase-1(ICE)和 caspase-3(CPP32)的X线结晶图像，结果显示它们具有相似的空间结构，在P20的C端和P10的N端有200多个氨基酸残基的序列尤为保守，在空间结构上组成相似的β折叠中心和相邻的α螺旋，保守的、对蛋白酶活性有特殊意义的氨基酸残基均位于这一段，并形成特定的结构。不同源的序列主要存在于P20的N 端和P20与P10交界处，这两个部位的氨基酸残基在蛋白酶活化过程中一般被全部或部分切除。 所有的成员都保守性地包含有与底物P1Asp作用的氨基酸残基，如在ICE中，它们是催化中心的Cys285，与酶/抑制剂复合物的巯基半缩醛以氢键结合的His237，以及可以稳定反应中间物氧阴离子的Gly238。另外，Arg179、Arg341、Gln383和Ser347形成容纳P1Asp的“口袋”，Ser339靠近Cys285的巯基，以氢键结合P1位的酰胺。与P2~P4作用的氨基酸残基则变异比较大，这可能是各个成员识别不同底物的特异性之所在。在活性Cys周围的氨基酸序列也很保守，一般都有 Gln-Ala-Cys-Arg-Gly(QACRG)五肽序列，在caspase-8、caspase-10中为QACQG，在caspase-9中是QACGG，这三个成员都有一个氨基酸残基的变异，但这种变异不影响蛋白酶的剪切活性和特异性。
新教材人教版必修第二册 5.1 基因突变和基因重组 学案
第5章基因突变及其他变异 第1节基因突变和基因重组 课堂互动探究案 课程标准 概述碱基的替换、增添或缺失会引发基因中碱基序列的改变阐明基因中碱基序列的改变有可能导致它所编码的蛋白质 及相应的细胞功能发生变化，甚至带来致命的后果 描述细胞在某些化学物质、射线以及病毒的作用下，基因突变概率可能提高，而某些基因突变能导致细胞分裂失控，甚至发生癌变 阐明进行有性生殖的生物在减数分裂过程中，染色体所发生的自由组合和交叉互换，会导致控制不同性状的基因重组，从而使子代出现变异 素养达成 1．阐明基因突变导致遗传物质的改变，可能引起生物性状的改变。认同基因突变为生物进化提供原材料的观念。(生命观念) 2．通过归纳与概括总结基因突变的特点与意义。(科学思维) 3．结合减数分裂的过程图解，理解基因重组的类型。(科学思维) 4．举例说明基因突变和基因重组在育种、人类遗传病等方面的应用，运用基因突变的原理解释一些变异现象。(社会责任)。 大熊猫的正常颜色是黑白色，科学家在野外偶然发现了一只棕白色大熊猫，经研究发现是由于基因突变引起的。请思考下列问题： (1)什么是基因突变？ (2)引起基因突变的因素是什么？ (3)基因突变的特点是什么？
探究点一基因突变 【师问导学】 一、基因突变的实例 1．阅读教材P80－81实例内容，探讨下列问题： (1)基因突变一般发生在细胞分裂的什么时期？ (2)结合DNA分子的结构特点和复制过程，分析DNA分子复制时容易发生基因突变的原因。 (3)产生镰状细胞贫血的根本原因是什么？变化后导致血红蛋白基因中碱基种类、数量和排列顺序发生了怎样的变化？ (4)根据上述资料分析可知，基因突变导致基因结构的改变，这种改变具体表现在哪些方面？ 2．基因突变一定会改变遗传信息和生物性状吗？试分析原因。 二、基因突变的原因和特点 1．癌变的原因是由于细胞内抑癌基因和原癌基因发生突变，癌细胞的特点之一是能进行无限增殖，医学上通常使用一定量的化学药剂对癌症病人进行化疗。另一方面接受化疗后的病人身体非常虚弱。 结合基因突变分析并回答下列问题： (1)化疗能够治疗癌症的原理是什么？
Caspase 3活性检测
Caspase 3 活性检测试剂盒 产品组成： 产品组成 BB-4106-1 BB-4106-2 BB-4106-3 规格 20 assays 50 assays 100 assays 裂解缓冲液 5ml 10ml 15ml Ac-DEVD-p NA 200ul 500ul 1ml 检测缓冲液 5ml 10ml 15ml DTT 100ul 150ul 250ul 产品简介： Caspase 3 活性检测试剂盒(Caspase 3 Activity Assay Kit)是采用分光光度法检测细胞或组织裂解液中Caspase 3酶活性或纯化的Caspase 3酶活性的试剂盒。 Caspase(Cysteine-requiring Aspartate Protease)是一个在细胞凋亡过程中起重要作用的蛋白酶家族。Caspase 3也称CPP32、Yama或apopain，有时被写作caspase-3或caspase3，属于caspase家族的CED-3亚家族(CED-3 subfamily)，是细胞凋亡过程中的一个关键酶。Caspase 3是哺乳动物细胞中研究最多的一个caspase。Caspase-3在正常状态下以酶原的形式存在于胞浆中，没有活性；但在细胞发生凋亡阶段，Caspase-3被激活，活化的Caspase-3由两个大亚基和两个小亚基组成，裂解相应的胞浆胞核底物，最终导致细胞凋亡。Caspase 3可以剪切procaspase 3、6、7和9，并可以直接特异性剪切许多caspase 底物，包括PARP，ICAD，gelsolin和fodrin等。这些由caspase 3介导的蛋白剪切是细胞凋亡分子机制的重要组成部分。另外，caspase 3在细胞核凋亡过程中也起到了关键作用，包括染色质固缩，DNA片段化等。同时caspase 3对细胞起泡也起到关键作用。 本Caspase 3 活性检测试剂盒是基于casepase 3可以催化底物Ac-DEVD-pNA产生黄色的pNA，pNA在405nm附近有强吸收，从而可以通过测定吸光度来检测caspase 3的活性。 本试剂盒用酶标仪检测或容量不超过100μl的分光光度检测杯检测，可用于培养细胞或新鲜组织样本的caspase-3检测。 使用方法： 一、裂解缓冲液和检测缓冲液配制 根据待测样品数准备裂解缓冲液和检测缓冲液，每1ml缓冲液中加入10ul DTT。 二、样品处理 A．细胞样品 1.收集2-5×106个细胞，在4℃，500×g条件下离心2-3分钟，小心吸取培养基， 尽可能吸干，收集细胞。 2.用冷PBS洗涤细胞两次，每次洗涤后尽可能吸干上清。 3.在细胞中加入100μl冷的裂解缓冲液，高速涡旋振荡15秒。 4.置冰上15分钟，每隔5分钟高速涡旋振荡15秒。 5.在4℃，500×g条件下离心5分钟。 6.快速将上清吸入另一预冷的干净离心管，置于冰上或-80℃冰箱保存备用。 B．组织样品
中性蛋白酶
1.1中性蛋白酶的来源 蛋白酶是一类催化蛋白质肽键，生成蛋白胨、蛋白肽及氨基酸等产物的水解酶，其广泛分布于自然界的植物、动物和微生物中。例如木瓜蛋白酶主要来自于植物木瓜，胰蛋白酶来自动物的胰腺，来自微生物的蛋白酶没有特定名称，例如有来自AS1.398枯草芽孢杆菌的蛋白酶，有来自宇佐美曲霉的蛋白酶等等，其中微生物来源的蛋白酶数量与种类最多，也最具研究、开发与生产价值。随着水解条件之一的pH值的升高，蛋白酶分为酸性蛋白酶、中性蛋白酶和碱性蛋白酶，这也同时划分了它们应用的领域有所不同。 1.2中性蛋白酶的研究现状 对于中性蛋白酶的研究主要集中于以下几个方面： 一是继续研究发现新的蛋白酶品种，为蛋白酶家族添加新成员，尽管此项工作的难度越来越大，但其意义不容否认。 二是对现有蛋白酶进行修饰、改性，延长和强化其功能，期望在降低应用成本的同时，减少酶本身的一些缺陷对其应用的限制。 三是通过生物技术手段提高酶产量，降低酶生产成本。现代生物技术不仅发达而且发展很快，技术应用的可选择范围也很广，例如物理或化学诱变技术、细胞质融合技术、转基因技术以及克隆技术等等。可以肯定和确认的是：上述技术的应用都取得了不同程度的效果；同时这些技术各有优势，所以并存至今。 四是提取和纯化技术方面，朱建星利用萃取技术使酶液浓度提高到发酵酶液的3.6倍多。刘东旺等应用盐析、层析和凝胶过滤等技术，纯化后的酶活力超过纯化前的19倍之多等等。此外，在纯化倍数提高的同时往往伴随着提取率的提高。 1.3中性蛋白酶的生产现状 中性蛋白酶的生产水平随着研究水平的提高而提高。总体上发达国家高于国内，而国内厂家之间也参差不齐，以微生物发酵来源的中性蛋白酶为例，报道说的范围从数百至一万多（u/ml）都有。导致这种差别的原因可能是不同类的菌种，不同来源的同类菌种，不同的菌株改良程度，不同的发酵工艺优化水平等等。 1.4中性蛋白酶的应用 蛋白酶在工业化应用酶中占的比例很大（超过50%），而中性蛋白酶在蛋白酶中占的比例也很大。占比大的原因之一是应用范围大而广。以中性蛋白酶为例，它可用于皮革业提高皮革的质量；可用于牙膏帮助清除牙渍；可用于饲料以提高消化吸收率；用于食品加工最为广泛，加入待焙烤的面团中可以调节面团筋力，加入肉类中可以使肉制品嫩化、改善口感，加入牛奶中可以加速凝乳，加入以蛋白质为主的废弃料中可以制取多种水解产品。 在蛋白酶应用中有些问题令人困惑不解值得重视，例如不同来源的蛋白酶水解蛋白质的主要位点有差别，所以即使使用同一种蛋白质原料，不同来源蛋白酶的水解产物和水解率也会有所区别；如果混合食用不同来源的蛋白酶，假如总酶量与单一蛋白酶相同，由于水解的主要位点增加，也有可能提高水解率和水解程度。 另外用于食品水解的蛋白酶还有一层要求就是风味和口味，目前主要问题是口味即苦味。尽管“风味蛋白酶”的诞生在一定程度上降低了疏水基团的外露，很大程度上减少了水解产物的苦味，不过因其高昂的价格和苦味的残留依然不能得心应手地应用。 1.5本课题的研究目的与意义 从上述内容可知，虽然蛋白酶的研究、生产与应用已相对成熟，但是还有一些问题值得研究和解决，包括为提高产酶量而进行的菌种特性的改善、发酵工艺的优化、发酵酶液的浓缩与纯化等等。其中最基础的就是菌种产酶能力的提高。 尽管前面提到各种改善菌种的现代生物技术，包括物理或化学诱变技术、细胞质融合技术、转基因技术以及克隆技术等等，有的已经相当先进。但是从成本性、发酵副产物的安全性与稳定性、实用技术的可推广性等方面考虑，对菌种进行诱变不失为行之有效和有研究价值的研究。虽然诱变
高一生物《基因突变和基因重组》知识点归纳
高一生物《基因突变和基因重组》知识点归纳 名词： 1、基因突变：是指基因结构的改变，包括DNA碱基对的增添、缺失或改变。 2、基因重组：是指控制不同性状的基因的重新组合。 3、自然突变：有些突变是自然发生的，这叫～。 4、诱发突变(人工诱变)：有些突变是在人为条件下产生的，这叫～。是指利用物理的、化学的因素来处理生物，使它发生基因突变。 5、不遗传的变异：环境因素引起的变异,遗传物质没有改变，不能进一步遗传给后代。 6、可遗传的变异：遗传物质所引起的变异。包括：基因突变、基因重组、染色体变异。 语句： 1、基因突变 ①类型：包括自然突变和诱发突变 ②特点：普遍性;随机性(基因突变可以发生在生物个体发育的任何时期和生物体的任何细胞。突变发生的时期越早，表现突变的部分越多，突变发生的时期越晚，表现突变的部分越少。);突变率低;多数有害;不定向性(一个基因可以向不同的方向发生突变，产生一个以上的等位基因。)。 ③意义：它是生物变异的根本来源，也为生物进化提供了最初的原材料。 ④原因：在一定的外界条件或者生物内部因素的作用下，使得DNA复制过程出现小小的差错，造成了基因中脱氧核苷酸排列顺序的改变，最终导致原来的基因变为它的等位基因。这种基因中包含的特定遗传信息的改变，就引起了生物性状的改变。
⑤实例：a、人类镰刀型贫血病的形成：控制血红蛋白的DNA上一个碱基对改变，使得该基因脱氧核苷酸的排列顺序—发生了改变，也就是基因结构改变了，最终控制血红蛋白的性状也会发生改变，所以红细胞就由圆饼状变为镰刀状了。b、正常山羊有时生下短腿“安康羊”、白化病、太空椒(利用宇宙空间强烈辐射而发生基因突变培育的新品种。)。 ⑥引起基因突变的因素：a、物理因素：主要是各种射线。b、化学因素：主要是各种能与DNA发生化学反应的化学物质。c、生物因素：主要是某些寄生在细胞内的病毒。 ⑦人工诱变在育种上的应用：a、诱变因素：物理因素---各种射线(辐射诱变)，激光(激光诱变);化学因素—秋水仙素等b、优点：提高突变率，变异性状稳定快，加速育种进程，大幅度地改良某些性状。c、缺点：诱发产生的突变，有利的个体往往不多，需处理大量的材料。d、如青霉素的生产。 2、基因突变是染色体的某一个位点上基因的改变，基因突变使一个基因变成它的等位基因，并且通常会引起一定的表现型变化。 3、基因重组： ①类型：基因自由组合(非同源染色体上的非等位基因)、基因交换(同源染色体上的非等位基因)。 ②意义：非常丰富(父本和母本遗传物质基础不同，自身杂合性越高，二者遗传物质基础相差越大，基因重组产生的差异可能性也就越大。);基因重组的变异必须通过有性生殖过程(减数分裂)实现。丰富多彩的变异形成了生物多样性的重要原因之一。 4、基因突变和基因重组的不同点：基因突变不同于基因重组，基因重组是基因的重新组合，产生了新的基因型，基因突变是基因结构的改变，产生了新的基因，产生出新的遗传物质。因此，基因突变是生物产生变异的根本原因，为进
《基因突变与基因重组》说课稿
《基因突变和基因重组》说课稿 一、教学背景分析。 1.教材内容、地位及学情分析 本节是人教版普通高中标准实验教科书生物必修2《遗传与进化》的第五章《基因突变及其他变异》的第一节内容。通过前面各章的学习，学生对“基因是什么”、“基因在哪里”和基因如何起作用“等问题已有了基本的认识。本章内容既是对前四章内容合乎逻辑的延续，又是学习第六章《从杂交育种到基因工程》和第七章《现代生物进化理论》的重要基础。 本节介绍了基因突变，从实例入手，通过对镰刀型细胞贫血症的分析，引入基因突变的概念，然后详细阐述基因突变的原因和特点、意义。本节内容引导学生从分子水平上理解遗传物质如何引起基因突变的。 学生对于生物变异的现象并不陌生，通过初中生物课的学习学生已初步认识到生物变异首先与遗传物质有关，其次与环境有关，本节内容在此基础上，进一步引导学生学习遗传物质究竟是如何引体生物变异 2.教学目标 1、知识目标： （1）举例说明基因突变的概念。 （2）举例说明基因突变的特点和原因。 （3）说出基因突变的意义。 2、能力目标： （1）通过对课本中实例的分析，培养学生分析归纳总结的逻辑推理能力。 （2）通过学生之间相互启发、相互补充、激发灵感，提高学生合作—探究的能力。 3、情感目标： （1）通过生物变异的事例，增强学生对生物世界探究的好奇心及保护意识，培养学生们严谨的科学态度和热爱科学的兴趣。 （2）引领学生进入“自主—合作—探究”新课程理念氛围，让学生真正成为学习的主人。 3.教学重点、难点 （1）教学重点 基因突变的概念、特点及原因。 （2）教学难点 基因突变的意义。 二、教学展开分析 1.教具准备 实物投影仪、电脑演示教学软件 2.课时安排 1课时 3.教学方法和手段 利用多媒体课件，创设形象生动的教学氛围；同时应用讲述法、谈话法、指导读书法