Role of Fas ligand and receptor in the mechanism of T-cell depletion in acquired immunodeficiency syndrome: effect on CD4+ lymphocyte depletion and human immunodeficiency virus replication

Direct killing of CD4+ lymphocytes by human immunodeficiency virus-1 (HIV-1) probably cannot account for the magnitude of the loss of these cells during the course of HIV-1 infection. Experimental evidence supports a pathophysiologic role of the apoptotic process in depletion of CD4 cells in acquired immunodeficiency syndrome (AIDS). The Fas-receptor/Fas-ligand (Fas-R/Fas-L) system mediates signals for apoptosis of susceptible lymphocytes and lympoblastoid cell lines. A number of investigators have recently reported increased expression of the Fas receptor in individuals with HIV infection, along with increased sensitivity of their lymphocytes to anti-Fas antibody mimicking Fas ligand. We attempted to determine the role of Fas-mediated apoptosis in disease progression and viral replication. Increased Fas-receptor (CD95) expression on CD4+ and CD8+ lymphocytes was found in a large group of HIV-1-infected patients compared with normal controls; individuals with a diagnosis of AIDS and a history of opportunistic infection had significantly more Fas receptor expression than did asymptomatic HIV-infected persons and normal blood donor controls (P < .01). Triggering of the Fas-R by agonistic anti-Fas monoclonal antibody, CH11, was preferentially associated with apoptosis in the CD4+ cells; this effect was more pronounced in lymphocytes derived from HIV+ individuals. Soluble and membrane-bound forms of Fas-L were produced in greater amounts in peripheral blood mononuclear cells (PBMC) cultures and in plasma obtained from HIV-1-infected persons than from normal controls. Furthermore, triggering of lymphocytes from HIV-infected persons by CH11 increased levels of interleukin-1beta converting enzyme (ICE), a protein associated with apoptosis. When PBMC were cultured in the presence of CH11, p24 production per number of viable cells was decreased as compared with the same PBMC without CH11 (P < .01). These findings suggest that multiple mechanisms, including increased production of Fas-L by infected PBMC, increased Fas-R expression, and induction of a protease of ICE family, may play roles in the apoptotic depletion of CD4+ cells in HIV infection.     

Induction of Bax-alpha precedes apoptosis in a human B lymphoma cell line: potential role of the bcl-2 gene family in surface IgM-mediated apoptosis

The members of the bcl-2 gene family are major regulators of programmed cell death, but their role in sIg-triggered apoptosis remains unclear. Using sensitive and resistant variants of the human B cell line BL-41, we studied the expression of the bcl-2 gene family during surface IgM-mediated apoptosis. We found constitutive Bcl-2 and Bcl-x expression, which remained unaltered after sIg cross-linking, in both resistant and sensitive cells. This and other experiments suggest that constitutive expression of Bcl-2 or Bcl-x alone is not sufficient to protect from activation-induced cell death in B cells. We therefore investigated Bax-alpha, the death-promoting splice variant of Bax, and found strong induction of both mRNA and protein upon sIg stimulation in sensitive cells. However, resistant subclones showed only weak expression, which was not inducible by sIg cross-linking. We provide evidence that up-regulation of Bax-alpha and the resulting imbalance of Bcl-2/Bax might be a major regulator of sIg-mediated apoptosis. Additionally, we found strong constitutive expression of Bcl-xs, the death promoting variant of Bcl-x, in sensitive cells, whereas resistant cells showed only weak Bcl-xs expression. Thus, we observed a much stronger expression of the death-promoting proteins Bax-alpha (inducible) and Bcl-xs (constitutive) in sensitive cells than in resistant cells. We therefore propose a potential role of the novel bcl-2 gene family members bcl-x and bax in surface IgM-triggered apoptosis.     

Bcl-2 is overexpressed and alters the threshold for apoptosis in a cholangiocarcinoma cell line

Cholangiocarcinoma is a malignant neoplasm originating from cholangiocytes. The mechanisms responsible for oncogenesis of cholangiocytes are unknown. Resistance to apoptosis, especially by altered expression of B-cell lymphoma/leukemia 2 (Bcl-2) family members, has been implicated as a mechanism contributing to malignant transformation. Thus, our aim was to test the hypothesis that altered expression of Bcl-2 family members by cholangiocarcinoma cells renders them resistant to apoptosis. We compared the apoptotic threshold and expression of the Bcl-2 protein family members, Bcl-2, Bcl-XL, and Bax, in two human cell lines: 1) nonmalignant human cholangiocytes immortalized by transfection with the simian virus 40 (SV 40) large T antigen; and 2) a malignant human cholangiocarcinoma cell line. Apoptosis was induced pharmacologically using beauvericin. Bcl-2, Bcl-x long, and Bax protein expression were evaluated by immunoblot analysis, and Bcl-2 expression was modulated using antisense technology. The cholangiocyte and malignant/nonmaligant phenotype of both cell lines was verified using both in vitro and in vivo approaches. Beauvericin induced apoptosis of nonmalignant cholangiocytes in a concentration- (0 to 25 micromol/L) and time- (0 to 6 hours) dependent manner. In contrast, malignant cholangiocytes were resistant to apoptosis. Although expression of Bcl-x long and Bax protein were similiar in the two cell lines, Bcl-2 protein expression was 15-fold greater in malignant than in nonmalignant cholangiocytes. An 18 mer bcl-2 antisense oligonucleotide reduced expression of Bcl-2 protein by 50% and increased the rate of beauvericin-induced apoptosis more than threefold in the malignant cells. Our results support the hypothesis that resistance to apoptosis by overexpression of Bcl-2 may be a feature of cholangiocarcinoma.     

Differential expression of cell survival and cell cycle regulatory proteins in cutaneous squamoproliferative lesions

Previous models of cutaneous carcinogenesis have primarily focused on the regulation of keratinocyte (KC) proliferation and differentiation. However, it has become clear in many neoplastic systems that altered rates of cell death and/or inability to undergo growth arrest can also contribute to the development of cancer. Apoptosis-regulatory proteins include those that block apoptosis such as Bcl-2 and Bcl-x, whilst a related protein Bax promotes apoptosis. Cell cycle regulatory proteins include those associated with growth arrest, i.e. p21wafl, p53, and those associated with proliferation, i.e. Ki-67. Paraffin embedded samples from ten different lesions of squamous cell carcinoma (SCC), Bowen's disease (BD), keratoacanthomas (KA), and nine normal adult skin samples were stained by immunohistochemistry to detect expression of Bcl-2, Bcl-x, Bax, Ki-67, p21wafl, p53 and apoptosis (TUNEL assay). Compared to low levels of Bcl-x and Bcl-2 immunostaining in normal skin, all the squamoproliferative lesions had strong and diffuse KC expression of Bcl-x (>80%) but minimal to absent KC Bcl-2 expression (<15%). Bax immunopositivity was limited to the basal layer in normal skin and BD. In contrast, by examining serial sections both Bcl-x and Bax appeared to be coexpressed by the majority of malignant KCs in KA and SCC (>70%). These immunostaining profiles reveal that squamoproliferative lesions, including invasive transformed KCs, preferentially express Bcl-x over Bcl-2, in addition to upregulating their Bax levels. Even though there were numerous TUNEL positive cells in these squamoproliferative lesions, no other evidence of apoptosis was seen reinforcing the necessity to use caution when relying on TUNEL staining for identification of programmed cell death in skin biopsies. Normal sun-exposed skin had low but detectable p53 and rare p21wafl KC expression. Significantly higher numbers of p21wafl and p53 immunopositive KCs were noted throughout the lesions in BD and SCC in contrast to KA where p53 and rare p21wafl immunopositive KCs were primarily limited to the periphery of the tumor cell islands. In general, p53 KC expression was higher in all squamoproliferative lesions and sun-exposed normal skin compared to p21Wafl expression. Summary of the expression of cell cycle regulatory proteins for both p21wafl and p53 KC expression was: SCC > BD > KA, in marked contrast to Ki-67 KC expression which was: BD > KA > SCC. The relatively few malignant cells in SCC that were actively participating in the cell cycle (i.e. Ki-67 positive) suggests that these neoplasms may arise primarily by increased cell survival and resistance to apoptosis rather than by hyperproliferation. These studies emphasize the importance of examining multiple members of protein families that regulate apoptosis, proliferation, growth arrest, and differentiation. It is the overall balance between these cellular phenomena that determine whether a cell remains viable or undergoes programmed cell death and contributes to the appearance of a neoplasm. The overexpression of Bcl-x may confer a survival advantage to malignant KCs unable to growth arrest to repair damaged DNA (mutant p53) and/or undergo terminal differentiation (increased p21wafl). Thus, mutation or aberrant expression of such proteins may participate in the multistep process of carcinogenesis that gives rise to these squamoproliferative lesions.     

Modulation of cell-cell adherens junctions by surface clustering of the N-cadherin cytoplasmic tail

PURPOSE: Testicular germ cell tumors (TGCTs) represent one of the few tumor types that are curable by antineoplastic therapy, probably due to the high sensitivity of this neoplasm to induction of apoptosis by chemotherapeutic agents and/or ionizing radiation. Here, we tested cell susceptibility to radiation-induced apoptosis in a panel of TGCT cell lines and attempted to correlate this with the known potentially relevant molecular determinants (p53 gene status and Bcl-2 family proteins) of apoptosis. METHODS AND MATERIALS: Induction of apoptosis by gamma-radiation was morphologically recognized in NT2, NCCIT, S2, and 2102 EP using Hoechst/PI staining and additionally confirmed by Western blot analysis of PARP cleavage. The p53 gene status was estimated by sequence analysis. Expression of p21/WAF/CIP was determined by Northern blot analysis and immunoblotting was used to monitor p53, Bax, Bcl-2, Bcl-x, and Bak protein levels. In vitro colony formation was studied to establish clonogenic survival curves. RESULTS: NT2 and NCCIT appeared to be susceptible for radiation-induced apoptosis, contrasting 2102 EP and S2 which were highly resistant. Sequence analysis showed that NT2, S2, and 2102 EP are homozygous for wild-type p53 (wtp53), whereas NCCIT contains mutant p53 (mtp53). NT2 and 2102 EP cells showed radiation-induced p53 upregulation, while NCCIT (mtp53) and S2 (no p53 protein) cells did not. Consistently, gamma-radiation-induced DNA damage resulted in a p53-dependent transactivation of the p21/WAF/CIP gene in NT2 and 2102 EP, but not in mtp53-containing NCCIT cells and p53 nonexpressing S2 cells. Constitutive expression of Bax, Bcl-2, Bcl-x, and Bak was not affected by radiation and showed no correlation with cell susceptibility to radiation-induced apoptosis. A discrepancy was found between apoptosis and reproductive death. CONCLUSIONS: The present study revealed that: i) the presence of wtp53 may not be absolutely required for the hypersensitivity for radiation-induced apoptosis in TGCT cell lines, ii) the molecular mechanism underlying the unique radiosensitivity was independent of the expression of Bcl-2 family proteins, and iii) cell susceptibility to apoptosis induction is not sufficiently informative to predict intrinsic radiosensitivity as determined by clonogenic survival.     

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.     

Nimodipine monotherapy and carbamazepine augmentation in patients with refractory recurrent affective illness

We investigated the expression and function of Fas and Fas ligand (FasL) on peripheral blood lymphocytes (PBLs). The cells were stimulated with various cytokines or 12-0-tetradecanoyl phorbol 13-acetate (PMA) plus ionomycin. About 30% of unstimulated PBLs expressed Fas, and the expression was augmented by interleukin-1beta (IL-1beta), IL-2, tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), or PMA plus ionomycin. Although only minimal FasL expression was detected on unstimulated PBLs, FasL expression was markedly induced by IL-2 or PMA plus ionomycin, suggesting that Fas and FasL were both expressed on IL-2-stimulated or PMA-plus-ionomycin-stimulated PBLs. Although IL-2-stimulated or PMA-plus-ionomycin-stimulated PBLs were positive for both Fas and FasL, no significant increase in apoptosis was demonstrated in these activated PBLs. In addition, treatment of PBLs with IL-2 or PMA plus ionomycin did not change anti-Fas-induced apoptosis, although these activated PBLs expressed Fas strongly when compared with unstimulated PBLs. Only IL-2-stimulated or PMA-plus-ionomycin-stimulated PBLs killed Fas+ target cells efficiently via the interaction of Fas on target cells with FasL of PBLs. Bcl-2 was constitutively expressed on unstimulated PBLs, but its expression was significantly augmented by IL-2 or PMA plus ionomycin. The expression of Bax was clearly induced only on IL-2-stimulated or PMA-plus-ionomycin-stimulated PBLs and that of other Bcl-2 family proteins such as Bcl-x and Bad could not be detected on human PBLs, including IL-2-stimulated or PMA-plus-ionomycin-stimulated PBLs. Our results suggest that PBLs activated by IL-2 or PMA plus ionomycin express both Fas and FasL and that they kill Fas+ target cells by using FasL on the surface. The resistance of these activated PBLs to Fas-mediated apoptosis may be due to the augmented Bcl-2 expression or the presence of Bcl-2:Bax heterodimers on these cells.     

Role of Akt and c-Jun N-terminal kinase 2 in apoptosis induced by interleukin-4 deprivation

We have shown previously that interleukin-4 (IL-4) protects TS1alphabeta cells from apoptosis, but very little is known about the mechanism by which IL-4 exerts this effect. We found that Akt activity, which is dependent on phosphatidylinositol 3 kinase, is reduced in IL-4-deprived TS1alphabeta cells. Overexpression of wild-type Akt or a constitutively active Akt mutant protects cells from IL-4 deprivation-induced apoptosis. Readdition of IL-4 before the commitment point is able to restore Akt activity. We also show expression and c-Jun N-terminal kinase 2 activation after IL-4 deprivation. Overexpression of the constitutively activated Akt mutant in IL-4-deprived cells correlates with inhibition of c-Jun N-terminal kinase 2 activity. Finally, TS1alphabeta survival is independent of Bcl-2, Bcl-x, or Bax.     

Apoptosis of lymphocytes induced by glucocorticoids and relationship to therapeutic efficacy in patients with systemic lupus erythematosus

OBJECTIVE: Glucocorticoids (GCs) are the drugs of first choice for treatment of systemic lupus erythematosus (SLE). However, the disease in some patients is resistant to these agents. This study evaluated the possibility of a relationship between response to GC treatment and the rate of apoptosis in vitro, and also analyzed Bcl-2 and Fas expression on peripheral blood mononuclear cells (PBMC) from patients with SLE in relation to GC-induced apoptosis. METHODS: Twenty-seven SLE patients and 13 normal controls were studied. Disease activity scores were determined before and after treatment and used to divide patients into 2 groups: GC-resistant and GC-responsive. Isolated PBMC were stimulated with anti-CD3 monoclonal antibodies, cultured with various concentrations of GC, and analyzed by alamar blue assay to determine the LC50, defined as the concentration of GC lethal to 50% of the cells. We measured the expression of CD4, CD8, Fas, and Bcl-2 by FACStar plus flow cytometry. RESULTS: Lymphocytes in the GC-resistant group of SLE patients showed a significantly lower percentage of GC-induced apoptotic cells than did lymphocytes from the responsive group or from normal controls. The LC50 in the resistant group was significantly higher than that in normal controls or the responsive group. The lymphocytes remaining in the resistant group after GC treatment were mainly CD8+, with a high expression of Bcl-2. There was no significant difference in Fas expression between the GC-responsive and GC-resistant groups. CONCLUSION: Determination of the LC50 may be useful in predicting the efficacy of GC treatment in SLE patients, and may be of use in considering other treatment options. CD8 and Bcl-2 double-positive lymphocytes that are insensitive to the apoptotic effect of GCs may play a role in the resistance to these agents in SLE patients.     

Bcl-x(L) can inhibit apoptosis in cells that have undergone Fas-induced protease activation

Programmed cell death or apoptosis provides an irreversible mechanism for the elimination of excess or damaged cells. Several recent studies have implicated the activation of the interleukin 1beta-converting enzyme/Ced-3 (ICE/Ced-3) family of proteases as the "point of no return" in apoptotic cell death, while others have suggested that loss of mitochondrial membrane potential (delta psi(m)) is the ultimate determinant of cell death. The temporal relationship of these two events during apoptosis and the role of Bcl-2 proteins in inhibiting these steps has not been defined. To examine these issues, control and Bcl-x(L)-transfected Jurkat T cells were treated with Fas antibodies in the presence and absence of the ICE protease inhibitor zVAD-FMK. ICE/Ced-3 protease activity was monitored by following the cleavage of poly(ADP-ribose) polymerase (PARP) and delta psi(m) was followed by rhodamine 123 fluorescence. Although Bcl-x(L) expression did not block Fas-induced protease activation, it substantially inhibited the subsequent loss of delta psi(m) and cell death in Fas-treated cells. In contrast, zVAD-FMK blocked PARP cleavage as well as loss of delta psi(m) and cell death. Together these data demonstrate that Bcl-x(L) can maintain cell viability by preventing the loss of mitochondrial membrane potential that occurs as a consequence of ICE/Ced-3 protease activation.     

Alteration of proteins regulating apoptosis, Bcl-2, Bcl-x, Bax, Bak, Bad, ICH-1 and CPP32, in Alzheimer's disease

Recently, apoptosis has been implicated in the selective neuronal loss of Alzheimer's disease (AD). Apoptosis is regulated by the B cell leukemia-2 gene product (Bcl-2) family (Bcl-2, Bcl-x, Bax, Bak and Bad) and the caspase family (ICH-1 and CPP32), with apoptosis being prevented by Bcl-2 and Bcl-x, and promoted by Bax, Bak, Bad, ICH-1 and CPP32. In the present study, we examined the levels of these proteins in the membranous and cytosolic fractions of temporal cortex in AD and control brain. In the membranous fraction, the levels of Bcl-2 alpha, Bcl-xL, Bcl-x beta, Bak and Bad were increased in AD. In the cytosolic fractions, the level of Bcl-x beta was increased, while Bcl-xL, Bax, Bak, and Bad and ICH-1L were unchanged. CPP32 was not detected in AD or control brain. These findings demonstrate a differential involvement of cell death-regulatory proteins in AD and suggest that Bak, Bad, Bcl-2 and Bcl-x are upregulated in AD brains.     

Peripheral V gamma 9/V delta 2 T cell deletion and anergy to nonpeptidic mycobacterial antigens in asymptomatic HIV-1-infected persons

Gamma delta T cells represent a minor population of human peripheral lymphocytes, the majority of them expressing the V delta 2/V gamma 9 TCR. Their accumulation in infectious disease lesions and their reactivity toward mycobacterial Ags suggest that V gamma 9/V delta 2 T cells play a role during infectious diseases. We have shown previously a significant expansion of the V delta 1 subset parallel to a dramatic decrease of the V delta 2 subset in PBMC from HIV-infected persons. To understand the mechanisms involved in the deletion of V delta 2 T cells, we analyzed their ability to respond in vitro to several V gamma 9/V delta 2 t cell-specific ligands. We observed that in 60% of asymptomatic HIV-infected persons, V delta 2 T cells exhibited a functional anergy to Daudi and to Mycobacterium tuberculosis stimulations. These observations were supported by the defective expansion of this subset to the recently described nonpeptidic phosphorylated Ag, TUBAg-1. Since V delta 2 responsiveness to mycobacterial Ags was shown to be normally dependent on IL-2 secretion by Th1-type CD4 T cells, the ability of IL-2 to restore V delta 2 T cells' responsiveness to TUBAg-1 was tested. V delta 2 T cell anergy persisted in spite of the presence of IL-2, and was frequently correlated with a defect in CD25 expression on stimulated V delta 2 T cells. Since V delta 2 anergy was associated with an in vivo depletion of this subset, we studied whether programmed cell death could be involved in this process, particularly because of their activated phenotype. Although peripheral V delta 2 T cells from some HIV-infected persons showed an increased susceptibility to spontaneous and activation-induced apoptosis, statistical comparison between HIV+ and HIV- donors indicated that there was no difference between both groups in the rate of V delta 2 apoptosis. Finally, V delta 2 complementarity-determining region 3 TCR analysis indicated that, in vivo, the remaining V delta 2 T cells were still polyclonal. All together these results suggest that the qualitative and quantitative alterations of the V delta 2 subset in the course of HIV infection are the consequence of a chronic antigenic stimulation, and raise the question of the contribution of a cellular ligand induced or modified by chronic HIV infection.     

Immune activation in HIV-infected African individuals. Italian-Ugandan AIDS cooperation program

OBJECTIVE: Immune activation induced by chronic infections, dietary limitations, and poor hygienic conditions is suggested to be present in African HIV infection and is at the basis of the hypothesis that HIV infection in Africa could be prevalently associated with immunopathogenetic mechanisms. Very limited data are nevertheless available supporting this theory, and in particular no data are reported on functional and phenotypic analyses performed on fresh peripheral blood mononuclear cells (PBMC) of African HIV-infected patients living in Africa. DESIGN: Immunological and virological parameters were analysed in fresh PBMC of HIV-infected African and Italian patients with advanced HIV disease and comparable CD4 and CD8 counts, sex, and age. Both functional (antigen- and mitogen-stimulated cytokine production) and phenotypic (activation markers; markers preferentially expressed by T helper (Th) type 2 cells or by memory and naive cells) analyses were performed. Results were compared with those of HIV-seronegative African and Italian controls. HIV plasma viraemia was analysed by competitive polymerase chain reaction (PCR) and branched DNA techniques. RESULTS: (1) The production of mitogen-stimulated IFN-gamma and TNF-alpha as well as the production of env peptide-stimulated IFN-gamma, TNF-alpha, and IL-10 are increased in African HIV infection; (2) the expression of activation and Th2-associated markers is augmented in African HIV infection as is the memory/naive ratio; (3) mitogen-stimulated IFN-gamma and IL-10 production, as well as the expression of activation and Th2-associated markers and the memory/naive ratio, are augmented in African compared with Italian controls; and (4) plasma viraemia is reduced in African compared with Italian HIV-infected individuals. CONCLUSIONS: These results, which are the first to be reported on fresh material from African HIV-infected patients living in Africa, indicate that HIV disease is associated with an abnormal immune hyperactivation and may be accompanied in these patients by lower loads of virus, and show that such activation is present even in HIV-seronegative controls.     

Short-term stability of HIV provirus levels in the peripheral blood of HIV-infected individuals

Changes in viral load have been reported to reflect disease progression or response to therapy; however, the stability of HIV DNA levels in HIV-infected individuals has not been extensively studied. Cellular HIV DNA levels in infected individuals were evaluated over a short time period to determine degree of variability as well as any correlation with other measurements of virus load or immune status. Peripheral blood mononuclear cells (PBMC) were obtained several times over 1 month from 32 asymptomatic or symptomatic non-AIDS, HIV-infected individuals currently on AZT therapy. PCR amplification of the HIV gag region was performed with DNA from PBMC lysates and the PCR amplified products quantitated by liquid phase hybridization. HIV DNA levels in the majority of the patients were relatively stable, with 26 of 32 persons having less than threefold change. Changes over the study period were both positive and negative, and the median change in HIV DNA levels was 68.6%. These changes were found to positively correlate with fluctuations in plasma p24 levels. In contrast, no correlations were found with other measurements of immune system activity, including changes in CD4 number, CD4 percent, and beta 2-microglobulin when compared with provirus changes. This study shows that levels of HIV DNA can be relatively stable over short periods in most non-AIDS, HIV-infected persons.     

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.     

Expression of pro- and anti-apoptosis gene products in brains from paediatric patients with HIV-1 encephalitis

We have previously demonstrated the presence of DNA fragmentation in neurons, macrophages and microglia consistent with apoptosis, but not in reactive astrocytes in brain tissue from paediatric patients with HIV-1 encephalitis (HIVE). To further understand the underlying mechanism(s) for these findings as they relate to gene-directed neural cell death, we studied the in-situ expression of the Bcl-2 family of proteins, including the pro-apoptosis gene product Bax, the anti-apoptosis gene product Bcl-2, and Bcl-x. We demonstrate significantly elevated numbers of Bax-positive microglia and macrophages immunoreactive in basal ganglia and cerebral cortex of children who had HIVE, in comparison to HIV-1 infected children without encephalitis or children who were seronegative for HIV-1. In contrast, patients with HIVE, but not HIV-1 without encephalitis, or seronegative controls, had increased expression of Bcl-2 and Bcl-x in reactive astrocytes in cortex and basal ganglia. In vitro studies using Western blot analysis demonstrated an up-regulation in the levels of Bax, and phosphorylated (i.e. inactive) Bcl-2 in HIV-1 infected macrophages, and in LPS-activated macrophages, relative to levels in virus-negative unstimulated macrophages. These results suggest that productive HIV-1 infection, or cellular activation, renders macrophages more vulnerable to apoptosis. Taken together, these findings suggest that brain-resident macrophages and microglia in patients with HIV-1 encephalitis are more prone to undergo apoptosis and that astrocytes in contrast may be resistant to apoptosis. This may represent a mechanism to limit microglial activation and the spread of productive HIV-1 infection in the CNS of children with HIV-1 encephalitis.     

Old and new substrates in clinical nutrition

Bcl-2 has been demonstrated to inhibit apoptosis in breast cancer cells in vitro, and the ratio between Bcl-2 and its proapoptotic homologue Bax seems to be an important determinant of cellular sensitivity to induction of apoptosis. However, little information is available on the relationship between Bcl-2 and the rate of apoptotic and necrotic cell death in breast tumours. From a series of 441 premenopausal, lymphnode-negative breast cancer patients, a subset of 49 tumours was selected in which immunostaining for the 26-kDa isoform of Bcl-2 was either absent (n = 23) or very high (n = 26). High expression of Bcl-2 was found to be strongly associated with low rates of apoptotic (P < 0.001) and necrotic cell death (P < 0.001). The mean value of the apoptotic index was 2.69%+/-1.40% in Bcl-2-negative tumours and 0.68%+/-1.00% in Bcl-2-positive tumours. Expression of the proapoptotic protein Bax correlated neither with Bcl-2 nor with the frequency of apoptotic cells. Immunostaining for the antiapoptotic Bcl-2 homologue BcI-X(L) correlated with Bcl-2 expression (P < 0.001) but not with apoptosis. High proliferation rate and high tumour grade (Bloom-Richardson) were strongly associated with absence of Bcl-2 expression (P< 0.001). p53 accumulation was associated with absence of Bcl-2 expression and increased apoptotic activity. Loss of Bcl-2 expression was strongly correlated with increased apoptotic and necrotic cell death, high proliferation rate and high tumour grade, supporting a model in which Bcl-2 not only mediates cell death, but also cell division in breast cancer tissue, and in which regulation of cell division and cell death are tightly linked.     

Apoptosis loss and bcl-2 expression: key determinants of lymph node metastases in T1 breast cancer

The Bcl-2 proto-oncogene extends cell survival but does not confer any proliferative advantage to cells that express it. Thus, the loss of apoptosis may have a role in progression allowing the acquisition of additional mutations. To determine whether apoptosis loss at diagnosis is associated with the metastatic advantage of ductal breast carcinomas and to examine the relationship between Bcl-2 expression, p53, and tumor cell death status, we examined tumor samples from 116 patients diagnosed with T1 (2 cm or less) breast cancer with (n = 49) or without (n = 67) lymph node metastases. Apoptosis loss in histological sections was considered when <1% of tumor nuclei were stained with terminal deoxynucleotidyl transferase labeled with biotin. We studied the expression of Bcl-2 and p53 by immunohistochemistry and in 37 p53 mutations by single-strand conformational polymorphism analysis and cycle sequencing. Multivariate logistic regression modeling was used to estimate prevalence odds ratios (pORs) for apoptosis loss and presence of lymph node metastases. Patients with marked apoptosis loss in their tumor cells were about 5 times more likely to present lymph node metastases than those with no apoptosis loss in their tumor cells (adjusted pOR, 4.7; 95% confidence interval, 1.4-15.6; trend test, P = 0.008). Bcl-2 expression was strongly associated with both apoptosis loss (pOR, 6.9; trend test, P < 0.0001) and presence of lymph node metastases (pOR, 5.7; trend test, P = 0.002). These associations were more evident in histological grade I and II tumors than in poorly differentiated histological grade III tumors and in p53-negative tumors than in p53-positive tumors. This study demonstrates for the first time that the lymphatic progression of T1 human breast cancer is strongly related to apoptosis loss.