Expression of p53, Bcl-2 and Bax in cisplatin-induced apoptosis in testicular germ cell tumour cell lines

We examined the sensitivity for cisplatin-induced apoptosis in a panel of four testicular germ cell tumour (TGCT) cell lines and monitored the cellular expression of the apoptosis-related proteins p53, Bcl-2 and Bax. Three of four TGCT cell lines (NT2, NCCIT and S2) were hypersensitive for cisplatin-induced apoptosis, while the TGCT cell line 2102 EP appeared to be resistant for cisplatin-induced apoptosis, even at relatively high drug concentrations (12.5 microM). For all four cell lines, the induction of apoptosis by cisplatin correlated with drug sensitivity in the MTT assay. The differences in chemosensitivity and induction of apoptosis could not be attributed to differences in cellular platinum accumulation, DNA platination or platinum-DNA adduct removal. We next analysed the relationship between p53 status and cisplatin-induced up-regulation of p53, and the susceptibility to cisplatin-induced apoptosis. Wild-type p53 containing NT2 and 2102 EP cells showed p53 up-regulation upon drug treatment, and NCCIT (mutant p53) and S2 (no p53 protein) cells did not. Consistently, the increase in wild-type p53 protein in NT2 and 2102 EP cells led to an increase in mRNA level of the p53 downstream gene p21/WAF/CIP, whereas mutant p53-containing NCCIT cells and p53-non-expressing S2 cells could not transactivate this p53-responsive gene. As NT2, NCCIT and S2 were readily triggered into apoptosis, while 2102 EP cells failed to undergo cisplatin-induced apoptosis, our data suggest that the presence of wild-type and/or transactivation-competent p53 might not be an absolute prerequisite for efficient induction of apoptosis in TGCT cell lines. Also endogenous levels of Bcl-2 and Bax expression did not correlate with cisplatin-induced apoptosis. In addition, the endogenous Bcl-2 and Bax expression was not affected by cisplatin treatment. The present study suggests that, at least in our panel of TGCT cell lines, hypersensitivity for cisplatin-induced apoptosis might not be necessarily correlated with the presence of wild-type p53 and is probably not associated with Bcl-2 and Bax expression.     

Sensitivity to Fas-mediated apoptosis in pediatric acute lymphoblastic leukemia is associated with a mutant p53 phenotype and absence of Bcl-2 expression

We investigated the antitumour effects of 1-(2,6-difluorophenyl)-1H,3H-thiazolo [3,4-a]benzimidazole (TBZ) a new anti-HIV-1 agent, on human promyelocytic HL60 leukaemia, both a parental and a multidrug resistant form (HL60R). HL60R overexpresses P-glycoprotein and, like HL60, lacks p53 protein expression. HL60 and HL60R show similar levels of Bcl-2 protein. In contrast to the conventional chemotherapeutic agents daunorubicin, etoposide and mitoxantrone, TBZ caused equal or even greater cytotoxicity in HL60R than in HL60, and this result was associated with a more marked induction of apoptosis in the drug resistant cells. The antitumour activity of TBZ occurred in the range of concentrations higher than those required to exert antiviral activity. TBZ seems to act in the presence of P-glycoprotein and Bcl-2 and in the absence of p53 and is able to circumvent the mechanisms of drug resistance and anti-apoptosis present in HL60R cells.     

Bcl-2 but not Bax expression is associated with apoptosis in normal and transformed squamous epithelium

Aberrant regulation of apoptosis may contribute to tumorigenesis. Relative levels of apoptosis regulatory proteins, such as Bcl-2 and Bax as well as interactions of these proteins with other gene products, may contribute to the rate of apoptosis in neoplasia. We examined Bcl-2 expression in 104 squamous cell carcinomas of the head and neck, as well as histologically normal mucosa several centimeters away from the tumor, and in control normal mucosa from patients without cancer. Immunohistochemistry and immunoblotting demonstrated Bcl-2 expression in 30% (31 of 104) of squamous cell carcinoma, with an increase in Bcl-2 protein levels compared with control normal mucosa from noncancer patients. Bcl-2-positive tumors demonstrated a 5-fold decrease in the number of apoptotic cells compared with Bcl-2-negative tumors. Bcl-2 protein expression was associated with poorly differentiated tumor grade but was not correlated with Bax expression or patient survival. These findings demonstrate that Bcl-2 contributes to apoptosis in normal and transformed squamous epithelium.     

p53, Bax and Bcl-2 expression, and apoptosis in gestational trophoblast of complete hydatidiform mole

This study investigates the extent of apoptosis in complete hydatidiform mole (CHM), using an in situ 3'-end DNA labelling (TUNEL) technique on formalin-fixed and paraffin-embedded sections. The sections were also immunostained with antibodies to p53, Bax and Bcl-2 proteins. In 10 normal placenta cases and 15 CHM cases, the apoptotic index was <1 and 2-4 per cent, respectively. The labelled trophoblastic cells possessed pyknotic nuclei and densely eosinophilic cytoplasm which corresponded well to the so-called apoptotic bodies by light and electron microscopy. The p53 positive reaction was restricted to the nuclei of cytotrophoblasts and intermediate trophoblasts, while the syncytiotrophoblasts showed only rare immunolocalization. Strong p53 expression was seen most often in cytotrophoblasts of CHM (>30 per cent of nuclei) which also showed a higher apoptosis index, while cytotrophoblasts in normal placentae were weakly and focally labelled (<10 per cent of nuclei). There were statistical differences between normal and CHM cases (P<0.05). Bcl-2 accumulation, on the other hand, was observed predominantly in syncytiotrophoblasts of normal placentae, and cytotrophoblasts and intermediate trophoblasts did not express Bcl-2 in all cases. Interestingly, syncytiotrophoblasts were found to be negative for Bax protein and positive in cytotrophoblast, which is consistent with the function of the protein in conveying increased apoptosis susceptibility to this cell population. The results show that the level of apoptosis correlates with the histological type of the gestational trophoblasts, and apoptosis index is higher in cytotrophoblasts in CHM. The fact that p53 quantitative expression and an increase in the Bax/Bcl-2 ratio were also observed in CHM suggested that they may contribute partly to the high level of apoptosis.     

Bax is an important determinant of chemosensitivity in pediatric tumor cell lines independent of Bcl-2 expression and p53 status

Susceptibility of a tumor cell to undergo chemotherapy-induced apoptosis appears to be dependent upon the balance of proapoptotic and survival factors that are expressed within any given cell. We have chosen to evaluate how expression of several of these proteins influences chemosensitivity of a panel of 10 pediatric tumor cell lines chosen from three tumor histiotypes: neuroblastoma, rhabdomyosarcoma, and pediatric glial tumors. The proteins evaluated were p53 and six members of the Bax/Bcl-2 family: three proapoptotic proteins (Bax, Bak, and Bcl-xS) and three survival factors (Bcl-2, Bcl-xL, and Mcl-1). We investigated whether there was any relationship between endogenous expression of these proteins and chemosensitivity (or resistance) to three chemotherapeutic agents that directly damage DNA (doxorubicin, actinomycin D, and topotecan) and a mitotic spindle poison (vincristine). Even though exogenous overexpression of wild-type p53 has been associated with a chemosensitive phenotype in several model systems we demonstrated no such relationship in these studies. In addition, expression levels of Bcl-2, Bcl-xL, Bcl-xS, Bak, or Mcl-1 did not correlate with sensitivity or resistance to the four drugs. However, there was a statistically significant correlation between endogenous levels of Bax protein and sensitivity to both doxorubicin and actinomycin D. We conclude that even though many proteins such as p53 and Bcl-2 have been shown to influence drug response when exogenously overexpressed in model systems, in unmodified cell lines endogenous protein levels may not generate the same results. We have demonstrated that endogenous Bax expression was the only protein found to be associated with chemosensitivity across the three different tumor histiotypes and propose that analysis of Bax may be a more useful prognostic indicator for tumor response to therapy than either p53 or Bcl-2.     

Apoptosis-related factors p53, Bcl2, and Bax in neuroendocrine lung tumors

Neuroendocrine (NE) lung tumors comprise four classes of progressive aggressiveness for which proliferation and apoptosis rates could both contribute to their distinctive behavior. As p53 mutations may favor escape from apoptosis through changes in Bcl2-Bax expression balance, which are survival and apoptotic genes, respectively, we studied 121 NE lung tumors (16 typical carcinoids (TC), 5 atypical carcinoids (AC), 29 large-cell NE carcinomas (LCNECs), and 71 small-cell lung carcinomas (SCLCs) using immunohistochemistry. We quantified apoptosis by terminal-deoxynucleotidyl-transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) in 31 of these cases. There was a significant increase of p53 mutant immunophenotype (defined as immunoreactivity with at least two antibodies for at least 20% of tumor cells) between atypical/typical carcinoids group and the LCNEC/SCLC group (P = 0.0003). There was an inverse correlation (P < 0.0001) between the scores of Bax and Bcl2 expression in individual tumors and a significant inversion of the Bcl2. Bax ratio between low-grade (typical and atypical carcinoids) and high-grade (LCNECs and SCLCs) tumors with a predominant Bax expression in the first group and predominant Bcl2 expression in the second. Whereas carcinoids had variable apoptotic indexes, LCNECs had high indexes (1.3 to 6.8%), Bcl2 overexpression, Bax down-regulation, and Bcl2.Bax ratio > 1 correlated with lower apoptotic index in both LCNEC and the pool of LCNECs and SCLCs (P < 0.05) and a lower survival rate in the group of atypical and typical carcinoids and LCNECs (P < 0.002). The highest levels of Bcl2 expression and Bcl2.Bax ratios were associated with p53 mutant immunophenotype (P = 0.02). Our results suggest that aggressiveness in NE lung tumors could be linked, in addition to proliferation, to apoptosis-related factors.     

Mutation of p53 gene and genomic instability in testicular tumors

BACKGROUND: During the past decade, studies of human cancer have begun to yield molecular information on the identify of the multiple genetic changes in the development and progression of tumorigenesis. We investigated alterations of p53 and genomic instability in testicular tumors. MATERIALS AND METHODS: Polymerase chain reaction (PCR) single-strand conformation polymorphism was performed for analysis from exons 5 to 8 of p53 gene in 22 cases and PCR-microsatellite instability analysis using 8 microsatellite markers were conducted in 19 cases of testicular tumor. RESULTS: No mutations were noted for exons 5 to 8 of the p53 gene. Differences in unrelated microsatellites for tumor and corresponding normal DNA were detected in 5 of 19 (26.3%) cases examined. Alterations noted in more than 2 microsatellites were observed in 3 of 19 (15.8%) and categorized as replication error (RER) phenotype. Two of 7 (28.6%) seminomatous and 1 of 12 (8.3%) non-seminomatous testicular tumors patients showed RER. Two of 16 (12.5%) stage T1-3N0M0 and 1 of 3 (33.3%) stage T1-3N1-3M0-1 showed RER. CONCLUSIONS: Alterations in microsatellite instability may be involved in the development of testicular tumor.     

Induction of apoptosis by the N-acetyl-galactosamine-specific toxic lectin from Viscum album L. is associated with a decrease of nuclear p53 and Bcl-2 proteins and induction of telomeric associations

Fluorescent probes serve as sensitive tools for obtaining structural and functional information in cellular systems. In spite of the high sensitivity provided by fluorescent reagents, cell surface receptors expressed in low numbers often escape detection with commonly used fluorescent probes. R-Phycoerythrin (R-PE), a molecule with a very high quantum yield, is often the reagent of choice for the detection of such low abundance events. We have developed streptavidin conjugates of two highly fluorescent 35-40 nm diameter polystyrene nanospheres, the green fluorescent FluoSpheres (Ex/Em 505/515) and red fluorescent TransFluoSpheres (Ex/Em 488/645). Like R-PE, the new reagents have peak excitations near 488 nm but differ in their emission maxima; 515 nm for the green nanospheres, 645 nm for the red nanospheres and 575 nm for R-PE. Hence the nanospheres are detected by flow cytometry in channels capable of detecting green (FL1) and red (FL3) fluorescence, while R-PE is detected in channel FL2. These nanospheres were tested for the detection of the sparsely expressed epidermal growth factor receptor (EGFR) of NIH-3T3 cells and the densely expressed EGFR of A431 cells. Results indicate that the nanosphere reagents are more sensitive than fluorescein-streptavidin and at least comparable in sensitivity to R-PE-streptavidin. The simultaneous use of these nanospheres with R-PE was also studied by concurrent staining of the CD3 and CD4 receptors in JURKAT cells. Labeling of CD4 receptors with streptavidin nanospheres and CD3 receptors with the R-PE-anti-CD3 conjugate confirmed the suitability of using the new nanospheres in combination with R-PE in multicolor flow cytometry experiments. This paper thus describes the use of alternative tools with detection sensitivity comparable to that of R-PE, but detected in different channels than R-PE, permitting their simultaneous use with R-PE.     

Lack of correlation between cisplatin-induced apoptosis, p53 status and expression of Bcl-2 family proteins in testicular germ cell tumour cell lines

We investigated the role of p53 and of the Bcl-2 family proteins in the apoptotic response of a panel of testicular tumour cell lines (NT2, NCCIT, S2 and 2102 EP). The p53 gene status and the capacity of the p53 protein to transactivate the p21/WAF/CIP gene were determined, and we examined the correlation between p53 status and the susceptibility to cisplatin-induced apoptosis. In contrast to wild-type p53-containing NT2 and 2102 EP cells, NCCIT (mutant p53) and S2 (no p53 protein) cells were shown to be p53-transactivation defective. However, NCCIT and S2 cells with non-functional p53 were readily triggered into apoptosis by cisplatin, whereas p53-transactivation competent 2102 EP cells failed to undergo cisplatin-induced apoptosis. The defective apoptotic pathway in 2102 EP cells was reflected by a 4-fold decreased sensitivity to cisplatin in the MTT assay. We further demonstrated that the p53-independent differential cisplatin sensitivity among the testicular germ cell tumour (TGCT) cell lines was not due to differences in cellular cisplatin accumulation or DNA platination. The pattern of endogenous expression levels of Bax, Bcl-2, Bcl-x and Bak, which was not modulated by cisplatin treatment, demonstrated that these Bcl-2 family proteins are not involved in drug-induced apoptosis in the TGCT cell lines. Our results suggest a lack of correlation between cisplatin-induced apoptosis, p53 status and expression of Bcl-2 family proteins in our panel of TGCT cell lines. We conclude that the cisplatin-induced apoptotic pathway in TGCT cell lines might be p53-independent and is probably not associated with differences in the Bcl-2/Bax rheostat.     

The trophic action of IL-7 on pro-T cells: inhibition of apoptosis of pro-T1, -T2, and -T3 cells correlates with Bcl-2 and Bax levels and is independent of Fas and p53 pathways

Signals from the IL-7R are essential for normal thymocyte development. We isolated thymocytes from early developmental stages and observed that suspensions of pro-T1, -T2, and -T3 cells rapidly died in culture. Addition of IL-7 promoted their survival, but did not induce cell division. Pro-T4 cells did not undergo rapid cell death, and their survival was therefore independent of IL-7. Death in the absence of IL-7 showed the hallmarks of apoptosis, including DNA fragmentation and annexin V binding; however, caspase inhibitors blocked DNA fragmentation, but did not block cell death. The trophic effect of IL-7 was partially inhibited by blocking protein synthesis. The p53 pathway was not involved in this death pathway, since pro-T cells from p53-/- mice also underwent cell death in the absence of IL-7. The Fas/Fas ligand pathway was not involved in cell death, since Fas-deficient pro-T cells died normally in the absence of IL-7, anti-Fas Abs did not protect cells from death in the absence of IL-7, and Fas expression was undetectable on cells at these stages. The IL-7 trophic affect correlated with increased intracellular levels of Bcl-2 and decreased levels of Bax, whereas no Bcl-X(L), Bcl-w, or Bad was detectable. Thus, maintaining a favorable Bcl-2/Bax ratio may account for the trophic action of IL-7.     

Bcl-2 protein expression: association with p53 and prognosis in colorectal cancer

In myocardial SPECT perfusion imaging, reorientation algorithms from transaxial image planes are used to generate short- and long-axis views of myocardial tracer uptake. We performed phantom experiments with 201Tl to delineate how image reorientation affects the results of quantitative image analysis. METHODS: Thirty consecutive patient studies were analyzed to characterize the distribution of the angle of reorientation in a clinical setting. Short-axis SPECT images of a cardiac phantom with and without a 180 degrees cold-spot insert were reconstructed with three different backprojection filters (ramp, Metz and Butterworth) and reoriented through different angles ranging from 45 degrees to 89 degrees. Four interpolation algorithms were used to calculate from the transaxial images the pixel values of the reoriented images: (a) a simple interpolator that averages the pixel values of the eight neighboring pixels of the transaxial image; (b) a three-dimensional linear interpolator; (c) a hybrid interpolator that combines a two-dimensional linear in-plane with a one-dimensional cubic across-plane interpolation; and (d) a three-dimensional cubic convolution interpolator. Images were reoriented twice with opposite angles so that the original and the reoriented images could be directly compared. Circumferential profile analysis was applied to determine the root mean square error of corresponding profiles and the difference of the extent and the severity of perfusion defects. Single and multivariate analyses of variance (ANOVA) were used to compare the effects of the reorientation angle, the backprojection filter and the interpolation algorithm. RESULTS: In the clinical studies, the angle between the transaxial and reoriented images was 75 degrees +/- 10 degrees (s.d.). In 48 phantom experiments, multivariate ANOVA demonstrated that the backprojection filter and the interpolation algorithm significantly affect the circumferential profiles and the extent and severity of a perfusion defect (p < 0.05). In contrast, the angle of reorientation was not a significant factor (p = ns). By univariate analysis, the three-dimensional cubic interpolator was associated with significantly (p < 0.05) less error than the simple and three-dimensional linear algorithms. Relative computation times (simple interpolator = 100%) were 119% for the three-dimensional linear, 136% for the hybrid and 243% for the three-dimensional cubic interpolator. CONCLUSION: For quantitative analysis of myocardial SPECT perfusion images, a Metz filter for filtered backprojection in combination with a three-dimensional cubic convolution interpolation for image reorientation appears to offer improved accuracy.     

Expression of Apo-1/Fas (CD95), Bcl-2, Bax and Bcl-x in myeloma cell lines: relationship between responsiveness to anti-Fas mab and p53 functional status

The down-regulation of apoptosis may be an essential mechanism for tumour cell expansion in slowly proliferating tumours such as multiple myeloma. We studied eight myeloma cell lines for the presence of Bcl-2, which inhibits apoptosis, of Bax, which counteracts Bcl-2, of Bcl-x(L) and Bcl-x(S), which act in an anti- and pro-apoptotic fashion, respectively, and of Apo-1/Fas, which induces programmed cell death, when activated by the Apo-1/Fas ligand or the relevant monoclonal antibody (mab). All cell lines constitutively expressed homogenous amounts of Bcl-2, but displayed different amounts of Bax and Bcl-x proteins. The Apo-1/Fas antigen could be detected in seven out of eight myeloma lines, but expression levels varied considerably. The relative expression levels of Apo-1/Fas correlated with that of Bax, but not with that of Bcl-2 or Bcl-x subtypes. Furthermore, the effectiveness of the Apo-1/Fas mab was associated with the relative expression levels of the Apo-1/Fas and with that of the Bax antigen, but not with that of the Bcl-2 and Bcl-x antigens. We further showed that wild-type p53 function is not required for Apo-1/Fas-induced apoptosis, nor is it necessary for the expression of Bax or Apo-1/Fas antigens in myeloma. In conclusion, our results suggest a p53-independent co-regulation of Apo-1/Fas and Bax, as well as a role for Bax in Apo-1/Fas-induced apoptosis in myeloma.     

Bax and Bcl-2 expressions predict response to radiotherapy in human cervical cancer

PURPOSE: The ratio of Bcl-2 to Bax expression determines survival or death following an apoptotic stimulus. In order to establish a new predictor of the outcome of treatment for human cervical carcinoma, we investigated the relationship between the expressions of the Bax and Bcl-2 proteins and the response to radiotherapy after the administration of 10.8 Gy. METHODS: A total of 44 patients with histologically proven carcinoma of the uterine cervix, including three with recurrent cervical stump carcinomas, were treated with definitive radiotherapy. The presence of mutations in exons 5-8 of the p53 gene was analyzed by a single-strand conformation polymorphism analysis and DNA sequencing. RESULTS: Forty patients were found to have wild-type p53, and the remaining four had mutant p53. The Bax and Bcl-2 protein expressions prior to radiotherapy did not correlate with response and survival. However, the Bax and Bcl-2 protein expressions after radiotherapy correlated with both response and survival. Bax-positive tumors showed significantly better responses than the Bax-negative tumors after 10.8 Gy radiation (P = 0.0002). In contrast, the Bcl-2-positive tumors showed significantly poorer responses than the Bcl-2-negative tumors after radiation (P = 0.002). Increased Bax expression after the 10.8 Gy radiotherapy was found to be correlated with good survival (P = 0.04). In contrast, increased Bcl-2 expression after such radiotherapy was correlated with poor survival (P = 0.002). CONCLUSION: The levels of Bax and Bcl-2 expression after 10.8 Gy radiotherapy are useful prognostic markers in patients with human cervical carcinoma.     

Attenuation of p53 expression and Bax down-regulation during phorbol ester mediated inhibition of apoptosis

Sorcin is a 22 kDa calcium binding protein that is widely distributed in mammalian tissues, including brain, and is associated with the ryanodine receptor (RyR) family of intracellular calcium-release channels in the heart. To determine the cellular sites for potential central functions of sorcin, we examined the electron microscopic immunocytochemical localization of antipeptide antisera against sorcin and against cardiac and brain RyR in the rat caudate-putamen nucleus (CPN), one of the few regions expressing high levels of brain RyR. Sorcin-like immunoreactivity (S-LI) was detected in both neurons and glia by using immunoperoxidase and immunogold methods. Of 1,735 profiles containing immunogold-silver labeling for sorcin, almost 50% were dendrites and many of these dendrites were spiny. The remainder were mainly small axons, axon terminals, and, more rarely, glia. Furthermore, analysis of dually labeled tissue sections showed the presence of sorcin in many of the dendrites and some of the axonal and glial processes containing RyR. In dendrites, gold-silver deposits showing S-LI were prominently localized to saccules of smooth endoplasmic reticulum and mitochondria, both of which are known to store calcium. These labeled structures were located near the plasma membrane at sites postsynaptic to excitatory-type asymmetric junctions, as well as non-synaptic portions of the plasma membrane. In axons, S-LI was also often seen at extrasynaptic sites on, or near, the plasma membrane. We conclude that in the rat CPN, sorcin may act independently or, in conjunction with RyR, to modulate cytoplasmic release of calcium, mainly from smooth endoplasmic reticulum and/or mitochondria in neurons.     

Prostaglandin E2 induces apoptosis in resting immature and mature human lymphocytes: a c-Myc-dependent and Bcl-2-independent associated pathway

Prostaglandin E2 (PGE2) is a known negative regulator of T lymphocyte proliferation. Previously we have indirectly evidentiated the involvement of PGE2 in apoptosis of lymphocytes both in vitro and in vivo. We have evaluated a possible direct effect of PGE2 on apoptosis. To this end we have investigated the in vitro effects of PGE2 on cell death, and its possible correlation with c-Myc and Bcl-2 proteins. We used freshly isolated unstimulated human lymphocytes from neonatal thymus, cord blood and adult peripheral blood. PGE2 induced DNA fragmentation in both peripheral and cord blood at 10(-7) to 10(-5) M concentrations, even though this induction was delayed in peripheral blood with respect to cord blood. Apoptosis induced by PGE2 was always associated with a dose-dependent increase of cellular steady state c-Myc protein levels, whereas Bcl-2 protein levels were not substantially affected. Unstimulated thymocytes showed spontaneous DNA fragmentation that occurred earlier and at higher levels in PGE2-(10(-5) M) treated cells with respect to untreated controls. Also in these cells, PGE2 produced an early increase of c-Myc protein expression, although Bcl-2 protein levels remained unchanged. In conclusion, PGE2 induces apoptosis with different kinetics on immature and mature T cells: this induction is associated with the increase of c-Myc protein expression and seems to be independent from Bcl-2 regulation.