Caspases: their intracellular localization and translocation during apoptosis.

The activation of the caspase family of proteases has been detected in numerous cell systems and appears to function as a common pathway through which apoptotic mechanisms may operate. Caspases are synthesized as precursors (pro-caspases) and are converted into mature enzymes by apoptotic signals. The effects of caspases in apoptosis are accomplished by the cleavage of numerous proteins located in different intracellular compartments. In the present study we have addressed the question of the subcellular localization of different pro- and active caspases as well as several other proteins, such as Apaf-1, calpain and DFF, which also play important roles in the apoptotic process. We found that at least three pro-caspases (pro-caspases-2, -3 and -9) were present in both the mitochondrial and cytosolic fractions of untreated Jurkat T lymphocytes. Only pro-caspase-2 was found in the nuclear fraction. Pro-caspases-7 and -8 were found only in the cytosolic fraction. In apoptotic cells, caspases-3, -8 and -9 were present in the cytosolic fraction, whereas caspases-3 and -9 were also found in the mitochondrial fraction and caspase-7 in the microsomal fraction. Caspases-2 and -3 were present in the nuclear fraction. The selective localization of pro-caspases in different subcellular compartments may play an important, but yet unknown, role in their activation. The translocation of active caspases to other subcellular compartments appears to be critical for the development of the apoptotic process.     

Silencing of death receptor and caspase-8 expression in small cell lung carcinoma cell lines and tumors by DNA methylation

Small cell lung cancer cell lines were resistant to FasL and TRAIL-induced apoptosis, which could be explained by an absence of Fas and TRAIL-R1 mRNA expression and a deficiency of surface TRAIL-R2 protein. In addition, caspase-8 expression was absent, whereas FADD, FLIP and caspases-3, -7, -9 and -10 could be detected. Analysis of SCLC tumors revealed reduced levels of Fas, TRAIL-R1 and caspase-8 mRNA compared to non-small cell lung cancer (NSCLC) tumors. Methylation-specific PCR demonstrated methylation of CpG islands of the Fas, TRAIL-R1 and caspase-8 genes in SCLC cell lines and tumor samples, whereas NSCLC samples were not methylated. Cotreatment of SCLC cells with the demethylating agent 5'-aza-2-deoxycytidine and IFNgamma partially restored Fas, TRAIL-R1 and caspase-8 expression and increased sensitivity to FasL and TRAIL-induced death. These results suggest that SCLC cells are highly resistant to apoptosis mediated by death receptors and that this resistance can be reduced by a combination of demethylation and treatment with IFNgamma.     

Expression of inhibitor of apoptosis proteins in small- and non-small-cell lung carcinoma cells

Small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) cells both initiate apoptotic signaling, resulting in caspase activation, after treatment with anti-cancer agents. However, in contrast to SCLC cells, NSCLC cells do not fully execute apoptosis. The apoptotic process in NSCLC cells seems to be blocked downstream of caspase activation, thus the failure of NSCLC cells to execute apoptosis could result from inhibition of active caspases by inhibitor of apoptosis proteins (IAPs). Here we investigate the mRNA and protein expression of IAPs in a panel of SCLC and NSCLC cell lines. The NSCLC cell lines had a stronger cIAP-2 expression at both mRNA and protein levels, while the SCLC cell lines had a higher level of XIAP protein. Expression of cIAP-1, cIAP-2, and XIAP, the most potent caspase inhibitors, was further investigated in three lung carcinoma cell lines after treatment with 8 Gy of ionizing radiation or etoposide (VP16). In response to treatment, the level of IAPs was not altered in a way that explained the differences in cellular chemo- and radiosensitivity. The intracellular localization of IAPs was analyzed in untreated and treated lung cancer cells. Surprisingly, we found that cIAP-2 was mainly detected in the mitochondrial fraction, although the function of this protein in mitochondria is unknown. No major relocalization of IAPs was observed after treatment. Taken together, these results indicate that IAPs alone are not the main factor responsible for the resistance of NSCLC cells to treatment.     

Evodiamine Induces G2/M Arrest and Apoptosis via Mitochondrial and Endoplasmic Reticulum Pathways in H446 and H1688 Human Small-Cell Lung Cancer Cells

The goal of this study was to evaluate the ability of EVO to decrease cell viability and promote cell cycle arrest and apoptosis in small cell lung cancer (SCLC) cells. Lung cancer has the highest incidence and mortality rates among all cancers. Chemotherapy is the primary treatment for SCLC; however, the drugs that are currently used for SCLC are less effective than those used for non-small cell lung cancer (NSCLC). Therefore, it is necessary to develop new drugs to treat SCLC. In this study, the effects of evodiamine (EVO) on cell growth, cell cycle arrest and apoptosis were investigated in the human SCLC cell lines NCI-H446 and NCI-H1688. The results represent the first report that EVO can significantly inhibit the viability of both H446 and H1688 cells in dose- and time-dependent manners. EVO induced cell cycle arrest at G2/M phase, induced apoptosis by up-regulating the expression of caspase-12 and cytochrome C protein, and induced the expression of Bax mRNA and by down-regulating of the expression of Bcl-2 mRNA in both H446 and H1688 cells. However, there was no effect on the protein expression of caspase-8. Taken together, the inhibitory effects of EVO on the growth of H446 and H1688 cells might be attributable to G2/M arrest and subsequent apoptosis, through mitochondria-dependent and endoplasmic reticulum stress-induced pathways (intrinsic caspase-dependent pathways) but not through the death receptor-induced pathway (extrinsic caspase-dependent pathway). Our findings suggest that EVO is a promising novel and potent antitumor drug candidate for SCLC. Furthermore, the cell cycle, the mitochondria and the ER stress pathways are rational targets for the future development of an EVO delivery system to treat SCLC.     

Phenotypic alterations of small cell lung carcinoma induced by different levels of wild-type p53 expression

p53 induces both growth arrest and apoptosis in cancer cells. To clarify whether the level of p53 expression determines the response of small cell lung carcinoma (SCLC) cells, we assessed the effect of various p53 levels on a p53-null SCLC cell line, N417, using a tetracycline (Tc)-regulated inducible p53 expression system. Apoptosis was induced in SCLC cells with high p53 expression. Although low levels of p53 induced G1 arrest accompanied by p21 expression, cells with G1 arrest seemed to undergo apoptosis after further cultivation. Expression of exogenous p21 induced G1 arrest but not apoptosis in SCLC cells, suggesting that p53-mediated G1 arrest was induced through p21 expression. Moreover, high level of p53 expression down-regulated Bcl-2 expression in SCLC cells, while Bax was consistently expressed irrespective to the level of p53 expression. These results suggest that p53-mediated apoptosis and G1 arrest depend on level of p53 expression in SCLC cells and that the relative dominancy of Bax to Bcl-2 is involved in the induction of apoptosis by high level of p53 expression.     

Phenylbutyrate-induced apoptosis and differential expression of Bcl-2, Bax, p53 and Fas in human prostate cancer cell lines

OBJECTIVE: To assess the mechanisms of action of phenylbutyrate (PB), an investigational chemotherapeutic agent for prostate cancer (PCa), in apoptosis induction in PCa cell lines in vitro. STUDY DESIGN: We analyzed the differential expression of different apoptosis modulators, Bcl-2, Bax, p53 and Fas, for their potential role in PB-induced apoptosis using relative quantitative flow cytometry (FCM). Both androgen-dependent (LNCaP) and androgen-independent (C-4-2, PC-3-PF and DU145) human PCa cell lines were examined. RESULTS: PB induced apoptosis in PCa cell lines in a dose-dependent manner. Fifty percent apoptosis could be induced by 5-10 mM PB. Bcl-2 was down-regulated 30-75% and the Bax:Bcl-2 ratio elevated in apoptotic PCa cell lines regardless of their androgen dependency or p53 status. FCM revealed a heterogeneous stimulatory effect on the expression of Bax and Bcl-2 in PC3-PF cells at 0.5-2.5 mM PB. In a p53-positive cell line (DU145), p53 was repressed by 70% and Fas elevated sixfold with 10 mM PB. CONCLUSION: Our data show that PB-induced PCa apoptosis is associated with the relative repression of Bcl-2 and with up-regulation of Bax and Fas proteins and that this PB-induced apoptosis is independent of p53 and androgen-dependency status of PCa cell lines.     

N-terminal cleavage of bax by calpain generates a potent proapoptotic 18-kDa fragment that promotes bcl-2-independent cytochrome C release and apoptotic cell death

Upon apoptosis induction, the proapoptotic protein Bax is translocated from the cytosol to mitochondria, where it promotes release of cytochrome c, a caspase-activating protein. However, the molecular mechanisms by which Bax triggers cytochrome c release are unknown. Here we report that before the initiation of apoptotic execution by etoposide or staurosporin, an active calpain activity cleaves Bax at its N-terminus, generating a potent proapoptotic 18-kDa fragment (Bax/p18). Both the calpain-mediated Bax cleavage activity and the Bax/p18 fragment were found in the mitochondrial membrane-enriched fraction. Cleavage of Bax was followed by release of mitochondrial cytochrome c, activation of caspase-3, cleavage of poly(ADP-ribose) polymerase, and fragmentation of DNA. Unlike the full-length Bax, Bax/p18 did not interact with the antiapoptotic Bcl-2 protein in the mitochondrial fraction of drug-treated cells. Pretreatment with a specific calpain inhibitor calpeptin inhibited etoposide-induced calpain activation, Bax cleavage, cytochrome c release, and caspase-3 activation. In contrast, transfection of a cloned Bax/p18 cDNA into multiple human cancer cell lines targeted Bax/p18 to mitochondria, which was accompanied by release of cytochrome c and induction of caspase-3-mediated apoptosis that was not blocked by overexpression of Bcl-2 protein. Therefore, Bax/p18 has a cytochrome c-releasing activity that promotes cell death independent of Bcl-2. Finally, Bcl-2 overexpression inhibited etoposide-induced calpain activation, Bax cleavage, cytochrome c release, and apoptosis. Our results suggest that the mitochondrial calpain plays an essential role in apoptotic commitment by cleaving Bax and generating the Bax/p18 fragment, which in turn mediates cytochrome c release and initiates the apoptotic execution.     

Synthesis of Bcl-2 in response to anthracycline treatment may contribute to an apoptosis-resistant phenotype in leukemic cell lines.

BACKGROUND: Some forms of chemoresistance in leukemia may start from failure of tumour cells to successfully undergo apoptosis and Bcl-2 may play a role in this defect. Therefore, we evaluated the Bcl-2 content and synthesis in relation with the apoptotic potential in leukemic cell lines after anthracycline treatment. METHODS: U937, HL60, and K562 cells and their drug resistant (DR) variants were treated with varying concentrations of Idarubicin (IDA). Apoptosis was evaluated by fluorescence microscopy after acridine orange staining. Bcl-2 and Bax content were evaluated either by flow cytometry after indirect immunolabelling or by Western blot. RESULTS: High Bcl-2 contents were not related to a poor ability to undergo apoptosis in U937, HL60, K562 and their DR variants. IDA induced a concentration-dependent increase in Bcl-2 content in all cell lines as long as they do not perform apoptosis. Enhanced Bcl-2 expression was inhibited by cycloheximide, actinomycin D, or antisense oligonucleotide directed against bcl-2 mRNA. Bcl-2 expression was also increased in the resistant U937 variant after serum deprivation or C2-ceramide treatment. The synthesis of Bcl-2 led to an increased Bcl-2/Bax ratio solely in the cells with an apoptosis-resistance phenotype. CONCLUSIONS: These data suggest that exposure to IDA induces Bcl-2 expression in leukemic cell lines, and that this mechanism could contribute to apoptosis resistance and participate in the acquisition of chemoresistance. They also confirm that the evolution of the Bcl-2/Bax ratio reflects apoptotic ability better than the steady state level of Bcl-2 expression.     

Susceptibility to drug-induced apoptosis correlates with differential modulation of Bad, Bcl-2 and Bcl-xL protein levels

To define the responses of apoptotic regulatory proteins to different chemotherapeutic agents, we investigated the expression of Bcl-2 family gene products, the release of cytochrome c, and the activation of pro-caspase-3 during apoptosis induced by Taxol and Thiotepa, in the MCF-7 breast carcinoma and the HL-60 leukemia cell lines. The earliest event induced by drug exposure was increase in Bad protein levels, followed by Bcl-2 down-regulation, cytochrome c release, and Bcl-xL and Bax up-regulation. Bak accumulation was a late event. Activation of pro-caspase-3 and cleavage of Bcl-2 protein occurred in the HL-60 cells only, and followed the cytochrome c release. The overall responses were qualitatively similar in both cell types, but MCF-7 cells treated with Taxol showed a significant delay in apoptosis, correlating with early up-regulation of Bcl-2 and delayed release of cytochrome c. We conclude that Bad up-regulation is an early indicator of a cellular response that will lead to cell death, but may be modulated by survival mechanisms, which cumulatively govern the ultimate susceptibility to apoptosis.     

Oleanolic acid initiates apoptosis in non-small cell lung cancer cell lines and reduces metastasis of a B16F10 melanoma model in vivo

Background

Drug resistance, a process mediated by multiple mechanisms, is a critical determinant for treating lung cancer. The aim of this study is to determine if oleanolic acid (OA), a pentacyclic triterpene present in several plants, is able to circumvent the mechanisms of drug resistance present in non-small cell lung cancer (NSCLC) cell lines and to induce their death.

Principal Findings

OA decreased the cell viability of the NSCLC cell lines A459 and H460 despite the presence of active, multidrug-resistant (MDR) MRP1/ABCC1 proteins and the anti-apoptotic proteins Bcl-2 and survivin. These effects are due to apoptosis, as evidenced by the capacity of OA to induce fragmentation of DNA and activate caspase 3. Induction of NSCLC cell death by OA cannot be explained by inhibition of the MDR proteins, since treatment with triterpene had little or no effect on the activity or expression of MRP1. Moreover, treatment with OA had no effect on the expression of the anti-apoptotic protein Bcl-2, but increased the expression of the pro-apoptotic protein Bax, altering the Bcl-2/Bax balance towards a pro-apoptotic profile. OA also decreased the expression of the anti-apoptotic protein survivin. Furthermore, OA decreased the expression of the angiogenic vascular endothelial growth factor (VEGF) and decreased the development of melanoma-induced lung metastasis.

Conclusion

Our data provide a significant insight into the antitumoral and antimetastatic activity of OA in NSCLC and suggest that including OA in the NSCLC regimens may help to decrease the number of relapses and reduce the development of metastases.     

mRNA expression of Bcl-2, Bax, caspase-3 and -7 cannot be used as a marker for apoptosis in bovine blastocysts

At present the most widely used technique for apoptosis detection in embryos remains the in situ visualization of DNA fragmentation by terminal deoxynucleotidyl transferase-dUTP nick end labelling (TUNEL) assay although this technique may be prone to artefacts. The aim of the present study was to investigate if the mRNA expression of a set of genes involved in apoptosis (Bax, Bcl-2, caspase-3 and -7) at an earlier point in the apoptotic cascade could be a good marker for apoptosis in in vitro produced bovine embryos. After normalization to the geometric mean of three reference genes, GAPD, YWHAZ and SDHA, mRNA expression levels of Bax, Bcl-2, caspase-3 and -7 were compared in embryos treated with an apoptosis inducer, staurosporine and in non-treated embryos. None of the genes were differently expressed in treated in comparison with non-treated embryos. In conclusion, mRNA expression of Bax, Bcl-2, caspase-3 and-7 cannot be used as a reliable apoptosis detection method. Immunofluorescent staining of caspase-3 and -7 is a better choice where as for Bcl-2 no reliable and practicable alternative is available at the moment.     

Curcumin induces small cell lung cancer NCI-H446 cell apoptosis via the reactive oxygen species-mediated mitochondrial pathway and not the cell death receptor pathway

Curcumin (diferuloylmethane), an active component of the spice turmeric, induces apoptosis in several types of malignancies. However, little is known about its anticancer activity in small cell lung cancer (SCLC). SCLC represents a highly malignant and particularly aggressive form of cancer, with early and widespread metastases and a poor prognosis. In this study, we found that curcumin does not activate caspase-8 cleavage or alter the expression of apoptotic receptors FAS and TRAIL in NCI-H446 cells, suggesting that curcumin-induced apoptosis is not associated with death receptor-mediated pathways in these cells. Instead, curcumin caused apoptosis by increasing Bax expression while decreasing the expression of Bcl-2 and Bcl-xL. Curcumin induced a rapid decrease in mitochondrial membrane potential and the release of cytochrome c into the cytosol, followed by activation of caspase-9 and caspase-3. In addition, curcumin-induced apoptosis was accompanied by an increase of intracellular reactive oxygen species (ROS) level. These results indicated that a ROS-mediated mitochondrial pathway played an important role in the process of curcumin-induced apoptosis of human SCLC NCI-H446 cells.     

Apoptosis induced by butyrate is independent of Jak/STAT signaling in a fibrosarcoma cell line

The aim of this study was to evaluate the participation of the Jak-1 and STAT-1 proteins in sodium butyrate-induced apoptosis in 2C4 cells derived from human fibrosarcoma. Making use of Jak-1 or STAT-1 deficient cell lines, we demonstrated that the apoptotic process induced by butyrate is independent of the presence of these proteins. In addition, this work showed that, although the constitutive expression of pro-caspases-2 and -3 is reduced in STAT-1 cells, the activity of caspase-3 is preserved in both Jak-1 and STAT-1 deficient cells and is similar to that seen in 2C4 parental cells. In conclusion, we demonstrated that the absence of functionally active Jak-1 or STAT-1 protein directly affects the TNF-alpha-induced apoptosis, but does not alter the sodium butyrate-induced apoptosis in cells derived from human fibrosarcoma.     

Protection by Bcl-2 against apoptotic but not autophagic cell death after photodynamic therapy

Photodynamic therapy (PDT) induces apoptosis in many cell types. Recent reports identified autophagy as an alternative cell-death process following PDT. Here we investigated the occurrence of autophagy after PDT with the photosensitizer Pc 4 in human cancer cells that are deficient in the pro-apoptotic factor Bax (human prostate cancer DU145) or the apoptosis mediator caspase-3 (human breast cancer MCF-7v) and in apoptosis-competent cells (MCF-7c3 stably overexpressing human pro-caspase-3 and Chinese hamster ovary CHO 5A100). Further, each cell line was also studied with and without stably overexpressed Bcl-2. By electron microscopy and immunoblot analysis, autophagy was observed in all cells studied, whether or not they were capable of typical apoptosis or overexpressed Bcl-2. Bcl-2 overexpression protected against PDT-induced apoptosis and loss of clonogenicity in apoptosis-competent cells (MCF-7c3 and CHO); however, it did not protect against the development of autophagy or against loss of clonogenicity in apoptosis-deficient cells (MCF-7v and DU145). The results show that autophagy may be the dominant cell death pathway following PDT in cells that are incapable of undergoing normal apoptosis. In such cells, Bcl-2 does not protect against autophagic death.     

Bcl-2 siRNA Augments Taxol Mediated Apoptotic Death in Human Glioblastoma U138MG and U251MG Cells

The anti-neoplastic drug taxol binds to β-tubulin to prevent tumor cell division, promoting cell death. However, high dose taxol treatment may induce cell death in normal cells too. The anti-apoptotic molecule Bcl-2 is upregulated in many cancer cells to protect them from apoptosis. In the current study, we knocked down Bcl-2 expression using cognate siRNA during low-dose taxol treatment to induce apoptosis in two human glioblastoma U138MG and U251MG cell lines. The cells were treated with either 100 nM taxol or 100 nM Bcl-2 siRNA or both for 72 h. Immunofluorescent stainings for calpain and active caspase-3 showed increases in expression and co-localization of these proteases in apoptotic cells. Fluorometric assays demonstrated increases in intracellular free [Ca²⁺], calpain, and caspase-3 indicating augmentation of apoptosis. Western blotting demonstrated dramatic increases in the levels of Bax, Bak, tBid, active caspases, DNA fragmentation factor-40 (DFF40), cleaved fragments of lamin, fodrin, and poly(ADP-ribose) polymerase (PARP) during apoptosis. The events related to apoptosis were prominent more in combination therapy than in either treatment alone. Our current study demonstrated that Bcl-2 siRNA significantly augmented taxol mediated apoptosis in different human glioblastoma cells through induction of calpain and caspase proteolytic activities. Thus, combination of taxol and Bcl-2 siRNA offers a novel therapeutic strategy for controlling the malignant growth of human glioblastoma cells. Special issue article in honor of Dr. George DeVries.     

Bax expression and apoptotic cell death in Fanconi anaemia peripheral blood lymphocytes

OBJECTIVE: Deregulated apoptosis might be involved in some of the features of Fanconi anaemia (FA). The possibility that the pro-apoptotic Bax protein could be involved in an increased susceptibility to apoptosis in FA patients was investigated. MATERIALS AND METHODS: Intracellular Bax expression, Bcl-2 expression (an anti-apoptotic protein) and cell death were analysed in 26 FA peripheral blood lymphocyte samples. RESULTS: Most FA samples (69%) displayed increased levels of Bax and were more susceptible to both spontaneous apoptosis and mitogen activation-induced cell death. Two subgroups were identified: one presented elevated levels of Bax (n = 18), whereas the other (n = 8), had Bax levels lower than controls. Two subgroups based on Bcl-2 expression were also identified: one with normal and another with high Bcl-2 expression. No inverse correlation was found between Bcl-2 levels and Bax expression. A clear difference in susceptibility to induced cell death could be observed between control and FA samples. The best correlation was observed between high levels of Bax and mitogen-induced apoptosis of cells; these displayed characteristics of necrosis secondary to apoptosis, suggesting that the intrinsic apoptotic pathway was being activated. CONCLUSION: Despite increased susceptibility to cell death induction, there was no correlation between Bax levels, chromosome breakage, haematological parameters or androgen therapy. The importance of apoptosis and Bax expression in the clinical development of FA awaits clarification.     

Defective stress kinase and Bak activation in response to ionizing radiation but not cisplatin in a non-small cell lung carcinoma cell line

We have here examined ionizing radiation (IR)-induced apoptotic signaling in one IR-sensitive small cell lung carcinoma (SCLC) and one resistant non-small cell lung carcinoma (NSCLC) cell line, both harboring mutant p53. In the sensitive SCLC cell line, IR induced conformational modulation of Bak and Bax, mitochondrial depolarization, and nuclear fragmentation. These events were not observed in the IR-resistant NSCLC cell line. However, in the same cells, cisplatin, a DNA-damaging drug, induced Bak and Bax modulation, mitochondrial depolarization, and nuclear fragmentation. Pre-mitochondrial signaling events were examined in order to further characterize the differing IR response. In the SCLC cell line, IR-induced apoptotic signaling was found to involve a MEKK1-related pathway and activation of the stress-activated kinases JNK and p38. In comparison, the NSCLC cell line had higher basal levels of activity of JNK and p38, and IR treatment did not further activate these kinases. However, NSCLC cells were sensitive to Bak modulation and apoptosis induced by a kinase-active mutant of MEKK1. Together, the results delineate a mechanism of IR resistance in NSCLC cells and indicate that IR and cisplatin induce Bak modulation and apoptosis via different pathways.     

FPTB, a novel CA-4 derivative, induces cell apoptosis of human chondrosarcoma cells through mitochondrial dysfunction and endoplasmic reticulum stress pathways

Chondrosarcoma is a malignant primary bone tumor that responds poorly to both chemotherapy and radiation therapy. The aim of this study was to elucidate the mechanism of the novel Combretastatin A-4 derivative, 2-(furanyl)-5-(pyrrolidinyl)-1-(3,4,5-trimethoxybenzyl)benzoimidazole (FPTB)-induced human chondrosarcoma cells apoptosis. FPTB induced cell apoptosis in human chondrosarcoma cell line but not primary chondrocytes. FPTB induced up-regulation of Bax and Bak, down-regulation of Bcl-2 and Bcl-XL and dysfunction of mitochondria in chondrosarcoma. FPTB also triggered endoplasmic reticulum (ER) stress, as indicated by changes in cytosol-calcium levels. We found that FPTB increased glucose-regulated proteins (GRP)78 but not GRP94 expression. In addition, treatment of cells with FPTB induced calpain expression and activity. Transfection of cells with GRP78 or calpain siRNA reduced FPTB-mediated cell apoptosis. Therefore, FPTB-induced apoptosis in chondrosarcoma cells through the mitochondria dysfunction and involves caspase-9 and caspase-3-mediated mechanism. FPTB also induced cell death mediated by increasing ER stress, GPR78 activation, and Ca(2+) release, which subsequently triggers calpain, caspase-12 and caspase-3 activity, resulting in apoptosis.     

Expression of cell survival/death genes: Bcl-2 and Bax at the rate of colon cancer prognosis

The rate of tumour growth is dependent on the balance between proliferation and apoptosis at all stages of carcinogenesis. Apoptosis inhibition, in turn, depends partly on the balance between expression of two cell death regulatory genes, Bcl-2 and Bax. Colon cancer has long been associated with disturbances in apoptosis regulation. The aim of our study was to determine the expression levels of Bcl-2 and Bax mRNAs in 1 microg sample of total RNA obtained from normal colon and colon adenocarcinoma. This study was intended to evaluate possible differences in Bcl-2 and Bax mRNA levels at particular stages of colon adenocarcinoma classified according to Duke's system. The apoptotic frequency (represented by Bax mRNA copy number) was inversely proportional to the decrease of Bcl-2 gene expression. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) was performed to confirm apoptosis.