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Neoplastic events are marked by uncontrolled cell proliferation. One major focus of cancer research has been to identify treatments that reduce or inhibit cell growth. Over the years, various compounds, both naturally occurring and chemically synthesized, have been used to inhibit neoplastic cell proliferation. Two such oncostatic agents, melatonin and retinoic acid, have been shown to suppress the growth of hormone-responsive breast cancer. Currently, separate clinical protocols exist for the administration of retinoids and melatonin as adjuvant therapies for cancer. Using the oestrogen receptor (ER)-positive MCF-7 human breast tumour cell line, our laboratory has studied the effects of a sequential treatment regimen of melatonin followed by all-trans retinoic acid (atRA) on breast tumour cell proliferation in vitro. Incubation of hormonally responsive MCF-7 and T47D cells with melatonin (10(-9) M) followed 24 h later by atRA (10(-9) M) resulted in the complete cessation of cell growth as well as a reduction in the number of cells to below the initial plating density. This cytocidal effect is in contrast to the growth-suppressive effects seen with either hormone alone. This regimen of melatonin followed by atRA induced cytocidal effects on MCF-7 cells by activating pathways leading to apoptosis (programmed cell death) as evidenced by decreased ER and Bcl-2 and increased Bax and transforming growth factor beta 1 (TGF-beta1) expression. Apoptosis was reflected morphologically by an increase in the number of lysosomal bodies and perinuclear chromatin condensation, cytoplasmic blebbing and the presence of apoptotic bodies. The apoptotic effect of this sequential treatment with melatonin and atRA appears to be both cell and regimen specific as (a) ER-negative MDA-MB-231 and BT-20 breast tumour cells were unaffected, and (b) the simultaneous administration of melatonin and atRA was not associated with apoptosis in any of the breast cancer cell lines studied. Taken together, the results suggest that use of an appropriate regimen of melatonin and atRA should be considered for preclinical and clinical evaluation against ER-positive human breast cancer. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6

BACKGROUND & AIMS: Mutations of c-K-ras occur commonly in colonic neoplasms. The aim of this study was to determine how c-K-ras mutations alter the responses to the chemopreventive agent sulindac. METHODS: The parental rat intestinal cell line IEC-18 and c-K-ras-transformed derivatives were treated with sulindac sulfide. Cell cycle distribution was determined by flow-cytometric analysis (fluorescence-activated cell sorter), apoptosis by DNA fragmentation (laddering), flow cytometry, and microscopy, and changes in gene expression by immunoblotting. RESULTS: Sulindac sulfide inhibited cell growth and induced apoptosis in a time- and dose-dependent manner more rapidly in and at lower concentrations in parental cells than ras-transformed cells. Expression of the sulindac sulfide arrested cells in G0/G1, but cells entered apoptosis throughout the cell cycle. Proapoptotic protein Bak was relatively high in untreated parental cells and increased markedly after sulindac sulfide but was low in untreated ras-transformed cells and did not increase after sulindac sulfide. Expression of other Bcl-2 family members was unchanged after sulindac sulfide. However, sulindac sulfide reduced levels of cyclin D1 protein and cyclin E- and cyclin D1- associated kinase activity. CONCLUSIONS: c-K-ras-transformed enterocytes are relatively resistant to sulindac sulfide-induced growth inhibition and apoptosis, which may result from specific reduction of bak expression. (Gastroenterology 1997 Dec;113(6):1892-900)

Apoptosis is a fundamental mechanism implicated in normal development. One of the most spectacular developmental events involving apoptosis is tail regression during amphibian metamorphosis. We analyzed how thyroid hormone (3, 5, 3'-triiodothyronine, T3), the orchestrator of metamorphosis, affects expression and function of the proapoptotic gene Bax in the tail muscle of free-living Xenopus tadpoles. During natural metamorphosis Bax mRNA was expressed in tail muscles and was spatially correlated with apoptosis. Precocious treatment of tadpoles with T3 induced Bax expression and apoptosis. To verify that Bax expression was causally related to apoptosis we used a naked DNA gene transfer method to express Bax in the dorsal tail muscle. This induced apoptosis, and the process was exacerbated by T3 treatment. To determine whether T3 effects on Bax expression involved transcriptional regulation, we injected a Bax promoter sequence into dorsal and caudal tail muscles. In the dorsal muscle, T3 treatment did not affect transcription from the Bax promoter. However, in the caudal muscle, T3 treatment significantly increased Bax transcription. We conclude that T3-induced apoptosis in Xenopus tadpole tail muscles involves Bax-activating and Bax-synergis tic mechanisms. These programs are induced in spatially and temporally distinct manners.

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.

Patients with acute promyelocytic leukemia (APL) usually relapse after all-trans retinoic acid (RA) treatment because this therapy fails to eradicate the malignant clone. Our data showed that KH 1060 and other 20-epi vitamin D3 analogs alone were potent inhibitors of clonal growth of NB4 cells, an APL cell line (ED50, approximately 5 x 10(-11) M). The combination of KH 1060 and 9-cis-RA synergistically and irreversibly enhanced this effect. Neither KH 1060 nor 9-cis-RA (10(-6) M, 3 d) were strong inducers of differentiation of NB4 cells. However, 98% of the cells underwent differentiation to a mature phenotype with features of both granulocytes and monocytes after exposure to a combination of both compounds. Apoptosis only increased after incubation of NB4 cells with 9-cis-RA alone (28%) or with a combination of 9-cis-RA plus KH1060 (32%). Immunohistochemistry showed that the bcl-2 protein decreased from nearly 100% of the wild-type NB4 cells to 2% after incubation with a combination of KH 1060 and 9-cis-RA, and the bax protein increased from 50% of wild-type NB4 cells to 92% after culture with both analogs (5 x 10(-7) M, 3 d). Western blot analysis paralleled these results. Studies of APL cells from one untreated individual paralleled our results with NB4 cells. Taken together, the data demonstrated that nearly all of the NB4 cells can be irreversibly induced to differentiate terminally when exposed to the combination of KH 1060 and 9-cis-RA.

A novel resistant variant of murine P388 leukaemia, P388/SPR, was identified by de novo resistance to doxorubicin (DOX) in vivo. This mutant displayed a similar level of cross-resistance to etoposide (VP-16) and other topoisomerase II (topo II) inhibitors. Further analysis of the phenotype revealed a broad cross-resistance to vinca alkaloids, alkylating agents, antimetabolites, aphidicolin and UV light. Low-level expression of mdr1 and P-glycoprotein (P-gp), as well as a modest impairment of cellular drug accumulation and partial reversion of resistance to DOX and VP-16 by cyclosporine, confirmed a moderate role of P-gp in conferring drug resistance in P388/SPR cells. Consistent changes in neither topo II expression or activity nor glutathione metabolism could be detected. Induction of apoptosis was significantly reduced in P388/SPR cells, as indicated by minimal DNA fragmentation. Analysis of oncogenes regulating apoptotic cell death revealed a marked decrease of bcl-2 in combination with a moderate reduction of bax protein, but a striking overexpression of the long form of the bcl-X protein. Transfection of human bcl-X-L into P388 cells conferred drug resistance similar to that of P388/SPR cells. The data suggest that overexpression of bcl-X-L results in an unusual phenotype with broad cross-resistance to non-MDR-related cytotoxins in vitro, and provide an interesting example of spontaneous overexpression of another member of the bcl-2 gene family in cancer. Images Figure 2 Figure 3 Figure 4

A point mutation in the pleckstrin homology domain of the mouse Bruton's tyrosine kinase (btk) gene results in an X-linked immune defect, Xid, characterized by immunologic unresponsiveness to polymeric carbohydrate Ags. In Xid mice, B cells specific for phosphocholine (PC) do not develop in peripheral lymphoid tissues because they either fail to be positively selected from the marrow or they are clonally deleted via an Ag-driven, receptor-mediated process. Overexpression of the bcl-2 gene allows PC-specific B cells to survive and mature in Xid mukappa anti-PC transgenic mice, but PC-specific B cells are not rescued by bcl-2 in Xid mu-only transgenic mice. The failure of bcl-2 to rescue PC-specific B cells, in mu-only transgenic mice suggests that either it does not correct the btk defect in the Ag-driven selection process that occurs in pre-B cells and/or in very immature B cells or that a btk-dependent proliferative phase is required for the selection and amplification of the PC-specific B cells in mu-only transgenic mice. The rescue of PC-specific B cells in mukappa transgenic mice indicates that bcl-2 can alter receptor-mediated B cell selection at late stages in B cell development. The rescued PC-specific B cells in Xid male mice do not exhibit an altered proliferation profile in response to B cell-stimulating agents compared with B cells from unmanipulated Xid mice; thus, they fail to respond to soluble anti-mu, or PC-dextran, but they proliferate in response to PC, anti-mu, or anti-id conjugated to Sepharose.

Therapeutic agents used in the treatment of chronic lymphocytic leukemia (CLL) are capable of inducing apoptosis in some (but not all) patient isolates. It is not yet clear whether cells that are resistant to one agent will also be resistant to others, and the mechanisms contributing to differential apoptosis sensitivity are not known. Here we report that glucocorticoid hormone and a clinically relevant chemotherapy combination (fludarabine plus mitoxantrone) fail to induce apoptosis in four of 24 CLL patient isolates. Apoptosis resistance was associated with elevated BCL-2 and BAX expression. Interestingly, incubation in vitro led to down-regulation of BCL-2 expression in both apoptosis-sensitive and apoptosis-resistant cells, whereas parallel down-regulation of BAX occurred only in the resistant samples. Evaluation of nuclear endonuclease content indicated that all of the apoptosis-sensitive samples contained appreciable levels of activity, whereas the endonuclease was not detected in the four populations of resistant cells. Our results indicate that nuclear endonuclease activity represents an excellent prognostic indicator of CLL apoptosis sensitivity that may be controlled by differential BCL-2 family polypeptide expression and signals from the in vivo microenvironment.

Bcl-2, bcl-x, and bax genes code for proteins that affect the susceptibility of cells to apoptosis. In general, the expression of bcl-2 or bcl-x inhibits apoptosis while bax promotes apoptosis. We examined the levels of these proteins by immunoblotting in resting and activated T cells and in thymocytes. Bcl-2 and Bax proteins vary coordinately, but Bcl-x varies independently: Bcl-2 and Bax are higher in splenic T cells than in thymocytes, and their levels increase even more after T cell activation. In contrast, Bcl-x is almost undetectable in splenic T cells but is manyfold greater in thymocytes and in activated splenic T cells. When CTLL-2 cells or activated T cells are starved of IL (IL-2), the level of Bcl-x but not Bcl-2 protein drops before the onset of apoptosis. Stable transfection of either bcl-2 or bcl-x expression plasmids promotes the survival of CTLL-2 cells in the setting of IL-2 withdrawal. Over 70 to 90% of the transfected cells remain viable at 48 h after IL-2 withdrawal when all of the control transfected cells are apoptotic. These findings suggest that a decrease in Bcl-x protein levels precedes apoptosis after IL-2 withdrawal in T cells and that transfected bcl-2 promotes survival after IL-2 withdrawal by functionally masking this drop in Bcl-x.

The authors established apoptosis resistant COS-1, myeloma, hybridoma, and Friend leukemia cell lines by genetically engineering cells, aiming at more efficient protein production by cell culture. COS-1 cells, which are most widely used for eukariotic gene expression, were transfected with human bcl-2 gene. Both bcl-2 and mock transfected COS-1 cells were cultured at low (0.2%) serum concentration for 9 days. The final viable cell number of the bcl-2 transfected cells was ninefold of that of the mock transfectants. Both bcl-2 and mock transfectants were further transfected with the vector pcDNA-λ containing SV40 ori and immunoglobulin λ gene for transiently expressing λ protein. The bcl-2 expressing COS-1 cells produced more λ protein than the mock transfected COS-1 cells after 4 days posttransfection.Mouse myeloma p3-X63-Ag.8.653 cells, which are widely used as the partner for preparing hybridoma, and hybridoma 2E3 cells were transfected with human bcl-2 gene. Both bcl-2 transfected myeloma and hybridoma survived longer than the corresponding original cells in batch culture. The bcl-2 transfected 2E3 cells survived 2 to 4 four days longer in culture, producing 1.5- to 4-fold amount of antibody in comparison with the mock transfectants.Coexpression of bag-1 with bcl-2 improved survival of hybridoma 2E3 cells more than bcl-2 expression alone. The bag-1 and bcl-2 coexpressing cells produced more IgG than the the cells expressing bcl-2 alone.Apoptosis of Friend murine erythroleukemia(F-MEL) cells was suppressed with antisense c-jun expression. The antisense c-jun expressing cells survived 16 days at non-growth state.

Recent studies of transgenic mice have confirmed that clonal deletion is involved in the development of B cells. However, little is known about intercellular and intracellular molecular events regulating B cell clonal deletion. We investigated the role of bcl-2 and cytokines in the regulation of B cell clonal deletion using anti-IgM-induced growth arrest and apoptosis in immature B cell lines as a model. We show here that overexpression of Bcl-2 protein in stably transfected immature B cells partially inhibits anti-Ig M-induced apoptosis but does not affect growth arrest. Similarly, IL-5 has a strong inhibitory effect on anti-IgM-mediated apoptosis but has a weak inhibitory effect on growth arrest. Finally, although both bcl-2 overexpression and exogenous IL-5 cooperate with bacterial LPS to block apoptosis, bcl-2 overexpression and exogenous IL-5 have no additive inhibitory effect on anti-Ig induced apoptosis. These findings indicate that anti-IgM-induced apoptosis is independently regulated from growth arrest and is controlled by at least two independent pathways: One is regulated by either Bcl-2 protein or IL-5 and the other is regulated by LPS. Activation of both the bcl-2/IL-5 and LPS pathways is necessary for complete inhibition of apoptosis, and presumably, clonal selection of the immature B cells.

bcl-2 gene expression confers a survival advantage by preventing cells from entering apoptosis. In contrast to the previously described cytoplasmic localization of Bcl-2 in epithelial cells in vivo, in this study we have demonstrated, in a series of human epithelial cell lines, that Bcl-2 also localizes to mitotic nuclei. Both immunocytochemical and immunoelectron microscopical examinations localize this protein to nuclei and in particular to chromosomes. Nuclear Bcl-2 expression in these cell lines is correlated with the cell cycle. There is relatively strong expression during mitosis, most intense during prophase and metaphase, declining in telophase and then the protein becomes undetectable soon after separation of the two daughter cells. The expression and distribution of Bcl-2 is influenced by treatment with excessive thymidine. These results indicate that Bcl-2 may protect the cells from apoptosis occurring during mitosis and suggest a possible role for the protein in cell immortalization.

We investigated the effect of overexpression of a BCL-2 transgene on the irradiation survival of the interleukin (IL)-3-dependent 32D cl 3 cell line in vitro. The 32D-BCL-2 clonal subline was selected by transfection of 32D cl 3 cells with a BCL-2 and neor-containing plasmid vector selecting for G418 resistance. Cell line 32D-BCL-2 was IL-3-dependent and showed detectable BCL-2 protein expression by Western analysis of cell lysates. The cells expressing BCL-2 showed the characteristic block of apoptosis by DNA ladder analysis of cells following deprivation of IL-3, where a decrease in DNA fragmentation of 32D-BCL-2 compared to 32D cl 3 DNA was observed after 200 to 1,000 cGy irradiation. The 32D-BCL-2 cell line demonstrated increased radioresistance at a dose rate of 110 cGy/min with an n and D0 of 8.70 and 130 cGy, respectively, compared to 5.03 and 101 cGy for parent 32D cl 3 cells (P = 0.015 and 0.018, respectively). At the clinical low dose rate of 7.5 cGy/min, the n and D0 for 32D-BCL-2 were 2.51 and 178 cGy compared to 1.96 and 148 cGy for 32D cl 3 (P = 0.074 and 0.071, respectively). Thus, expression of a BCL-2 transgene increased the gamma-irradiation resistance of 32D cl 3 cells in vitro. These data are consistent with recently published evidence for a role of BCL-2 in reducing intracellular free oxygen radicals. These results may explain the unexpected survival of BCL-2-containing tumor cells in bone marrow transplant recipients after low-dose-rate total body irradiation. © Wiley-Liss, Inc.

Tolerance in T lymphocytes can result from clonal anergy, or paralysis, of Ag-specific T cells. To investigate the molecular mechanisms responsible for anergy, a system in which tolerance can be induced in vitro was employed. Anergy, as defined by long-lived nonresponsiveness to normal antigenic stimulation for IL-2 production, was produced in cloned murine CD4+ Th1 cells. Here we report that such anergic Th1 cells express constitutively reduced amounts of the protein tyrosine kinase p56lck and constitutively elevated levels of the protein tyrosine kinase p59fyn. Because protein tyrosine phosphorylation is known to be important for the normal induction of IL-2 synthesis, these results suggest that T cell anergy may be maintained, at least in part, by alterations in tyrosine phosphorylation signaling events.

To investigate the requirements for CD2 expression in the activation of T lymphocytes via the CD3-TCR complex, we produced and characterized a series of CD2-variants of the IL-2 producing Jurkat leukemia cell line, J32 (surface phenotype, CD2+, CD3+, CD28+). These mutants were derived by radiation and immunoselection, and were cloned under limiting dilution conditions. A total of 3 out of 30 of these mutants selectively lost the expression of both CD2 surface molecules and CD2 mRNA, and retained the expression of the CD3-TCR complex and the CD28 molecule. A mitogenic combination of anti-CD2 antibodies (9.6 + 9-1) failed to stimulate activation of these variants as measured by mobilization of intracellular Ca2+ and by IL-2 production. The CD2- mutants stimulated with anti-CD3 or anti-TCR mAb revealed an 8- to 32-fold decrease in IL-2 production and IL-2 mRNA accumulation as compared with the parental cells. No alteration of CD3-TCR-induced mobilization of intracellular Ca2+ was observed in the CD2- mutants. Reconstitution of CD2 expression by gene transfer in two J32 CD2- mutants restored IL-2 production and IL-2 mRNA accumulation in responses to both anti-CD2 and anti-CD3-TCR mAb. These results are the first direct demonstration of the requirement for CD2 molecules in optimizing IL-2 response in human T cells stimulated via CD3-TCR complex.

Human bcl-2 and bag-1 DNA were introduced into mouse hybridoma 2E3- O cells and expressed. The expression of bcl-2 in BCMGneo-bcl2 transfectants was confirmed by ELISA and that of bag-1 in pZeo-bag1 was confirmed by western blotting. In batch cultures, the over-expression of bcl-2 prolonged the culture period by 2 days and co-expression of bcl-2 and bag-1 prolonged the culture period by 3 days. The delayed increase in the dead cell number in culture of the bcl-2 and bag-1 cotransfectant indicated the additional antiapoptosis effect of bcl-2 and bag-1 cotransfection in comparison with the bcl-2 only transfection. The bcl-2 transfectants (2E3O-Bcl2) produced antibody twofold per batch culture in comparison with 2E3-O cells transfected with BCMGSneo (2E3O-Mock). Enhancement of this MoAb production was due to the improved survival of the cells and was not due to stimulation of antibody production rate per cell by Bcl-2 expression. And the bcl-2 and bag-1 co-transfectant (2E3O-Bcl2-BAG1) produced antibody approximately fourfold of 2E3O-Mock per batch culture. Enhancement of this MoAb production was due to the improved survival of the cells and was partly due to stimulation of MoAb production rate per cell in the non-growing phase by the cotransfection. The method to engineer hybridoma cells genetically with bcl-2 and bag-1 for increasing viability and productivity would be widely applied for improving antibody productivity of hybridoma cultures.

The balance between pro- and antiapoptotic proteins can determine cellular fate. In this regard, the Bcl-2 and IAP protein families have evolved as highly conserved regulators of cell death. A further testament to their critical roles in maintaining balance between cell life and death may be the increasing implication of Bcl-2 and TAP proteins in the pathologies of human diseases. Although much has been learned about these families of proteins, future studies of the Bcl-2 and IAP families are sure to hold more exciting discoveries and will continue to reveal new strategies for combating human diseases.

Chromosome 13q14 deletions constitute the most common genetic abnormality in chronic lymphocytic leukemia (CLL). To identify the putative tumor suppressor gene targeted by 13q14 genomic loss, we completely sequenced and characterized a segment of 790 kb at 13q14 spanning the minimal region of loss in CLL. Transcribed sequences in the region were identified through database homology searches and exon-prediction analysis. Two-hundred kb at the centromeric end of the sequence contain five CpG islands, three previously identified genes LEU5/RFP2, LEU2, and LEU1, seven of seven EST clusters composed of10 ESTs, and a large number of predicted exons. Homology searches against the mouse EST database have allowed us to identify a highly conserved alternative first exon of the LEU2 gene, giving rise to a novel transcript, ALT1 (GenBank accession no. AF380424), which originates within a G+C region in the vicinity of the D13S272 marker. Two novel 3' exons of LEU2 were also identified and are present in both LEU2 and ALT1 transcripts. However, we have not identified any mutations in leukemia cases, or alterations in expression of mRNAs in the region, that might directly implicate these mRNAs in the pathology of CLL. The centromeric end of the sequence, where all reported genes are located, contains twice the expected amount of ALU repeats, whereas the telomeric end is LINE1 rich and contains four LINE1 elements longer than 4 kb, including two full-length LINE1 sequences. This feature of the sequence may favor the occurrence of chromosomal rearrangements and may confer instability to the region, resulting in deletions that may inactivate an as yet unidentified tumor suppressor.

The small and large intestines differ in their expression profiles of Bcl-2 homologs. Intestinal segment-specific Bcl-2 homolog expression profiles are acquired as early as by mid-gestation (18-20 weeks) in man. In the present study, we examined the question whether such distinctions underlie segment-specific control mechanisms of intestinal cell survival. Using mid-gestation human jejunum and colon organotypic cultures, we analyzed the impact of growth factors (namely insulin; 10 microg/ml) and pharmacological compounds that inhibit signal transduction molecules/pathways (namely tyrosine kinases, Fak, P13-K/Akt, and MEK/Erk) on cell survival and Bcl-2 homolog expression (anti-apoptotic: Bcl-2, Bcl-X(L), Mcl-1; pro-apoptotic: Bax, Bak, Bad). The relative activation levels of p125Fak, p42Erk-2, and p57Akt were analyzed as well. Herein, we report that (1) the inhibition of signal transduction molecules/pathways revealed striking differences in their impact on cell survival in the jejunum and colon (e.g., the inhibition of p125Fak induced apoptosis with a significantly greater extent in the jejunum [approximately 43%] than in the colon [approximately 24%]); (2) sharp distinctions between the two segments were noted in the modulatory effects of the various treatments on Bcl-2 homolog steady-state levels (e.g., inhibition of tyrosine kinase activities in the jejunum down-regulated all anti-apoptotics analyzed while increasing Bax, whereas the same treatment in the colon down-regulated Bcl-X(L) only and increased all pro-apoptotics); and (3) in addition to their differential impact on cell survival and Bcl-2 homolog expression, the MEK/Erk and P13-K/Akt pathways were found to be distinctively regulated in the jejunum and colon mucosae (e.g., insulin in the jejunum increased p42Erk-2 activation without affecting that of p57Akt, whereas the same treatment in the colon decreased p42Erk-2 activation while increasing that of p57Akt). Altogether, these data show that intestinal cell survival is characterized by segment-specific susceptibilities to apoptosis, which are in turn linked with segmental distinctions in the involvement of signaling pathways and the regulation of Bcl-2 homolog steady-state levels. Therefore, these indicate that cell survival is subject to segment-specific control mechanisms along the proximal-distal axis of the intestine.

Recent studies have demonstrated that the downstream caspases, such as caspase 3, act as executors of the apoptotic cascade after traumatic brain injury (TBI) in vivo. However, little is known about the involvement of caspases in the initiation phase of apoptosis, and the interaction between these initiator caspases (e.g. caspase 8) and executor caspases after experimental brain injuries in vitro and in vivo. This study investigated the temporal expression and cell subtype distribution of procaspase 8 and cleaved caspase 8 p20 from 1 h to 14 days after cortical impact-induced TBI in rats. Caspase 8 messenger RNA levels, estimated by semiquantitaive RT-PCR, were elevated from 1 h to 72 h in the traumatized cortex. Western blotting revealed increased immunoreactivity for procaspase 8 and the proteolytically active subunit of caspase 8, p20, in the ipsilateral cortex from 6 to 72 h after injury, with a peak at 24 h after TBI. Similar to our previous studies, immunoreactivity for the p18 fragment of activated caspase 3 also increased in the current study from 6 to 72 h after TBI, but peaked at a later timepoint (48 h) as compared with proteolyzed caspase 8 p20. Immunohistologic examinations revealed increased expression of caspase 8 in neurons, astrocytes and oligodendrocytes. Assessment of DNA damage using TUNEL identified caspase 8- and caspase 3-immunopositive cells with apoptotic-like morphology in the cortex ipsilateral to the injury site, and immunohistochemical investigations of caspase 8 and activated caspase 3 revealed expression of both proteases in cortical layers 2-5 after TBI. Quantitative analysis revealed that the number of caspase 8 positive cells exceeds the number of caspase 3 expressing cells up to 24 h after impact injury. In contrast, no evidence of caspase 8 and caspase 3 activation was seen in the ipsilateral hippocampus, contralateral cortex and hippocampus up to 14 days after the impact. Our results provide the first evidence of caspase 8 activation after experimental TBI and suggest that this may occur in neurons, astrocytes and oligodendrocytes. Our findings also suggest a contributory role of caspase 8 activation to caspase 3 mediated apoptotic cell death after experimental TBI in vivo.

The adult small and large intestines display distinct expression profiles of Bcl-2 homologs, known regulators of apoptosis. This is thought to indicate that control mechanisms of intestinal apoptosis are gut segment-specific. Little is known on the expression of Bcl-2 homologs during gut development. In man, intestinal features and functions are acquired largely by mid-gestation (18-20 wks); the question whether segment-specific controls of intestinal apoptosis are also acquired early during development remains open. In the present study, we approached this by investigating the expression of six Bcl-2 homologs (Bcl-2, Bcl-XL, Mcl-1, Bax, Bak, Bad), and one nonhomologous associated molecule (Bag-1), during development of the human ileum and colon (12-20 wks of gestation). Beginning at 18 wks, we found that the epithelial localization of Bcl-2 homologs displayed differential patterns (or gradients) in both the ileum and colon; however, the patterns of some of the homologs differed between the two segments. For instance, Bag-1 and Bcl-2 exhibited crypt-villus decreasing gradients of expression in the ileum but not in the colon, whereas Mcl-1 displayed differing compartimentalizations between the two segments. Further analyses indicated that the steady-state expression levels of Bcl-2 homologs underwent modulations between 12 and 20 wks; however, the observed developmental profiles contrasted significantly between the two segments. For example, Bcl-2, Bag-1 and Bak levels increased in the colon, but the levels of these same homologs decreased in the ileum. Furthermore, by 18-20 wks, we found that the expression levels of each Bcl-2 homolog analyzed differed greatly between the ileum and colon. Altogether, these data indicate that the expression of Bcl-2 homologs is modulated differentially during human gut development in order to establish, by mid-gestation, distinct expression profiles for the small and large intestines. This in turn suggests that gut segment-specific control mechanisms of human intestinal apoptosis are acquired early during fetal life.

Bag1 is a regulator of heat shock protein 70 kDa (Hsp70/Hsc70) family proteins that interacts with steroid hormone receptors. Four isoforms of Bag1 have been recognized: Bag1, Bag1S, Bag1M (RAP46/HAP46), and Bag1L. Although Bag1L, Bag1M, and Bag1 can bind the androgen receptor (AR) in vitro, only Bag1L enhanced AR transcriptional activity. Bag1L was determined to be a nuclear protein by immunofluorescence microscopy, whereas Bag1, Bag1S, and Bag1M were predominantly cytoplasmic. Forced nuclear targeting of Bag1M, but not Bag1 or Bag1S, resulted in potent AR coactivation, indicating that Bag1M possesses the necessary structural features provided it is expressed within the nucleus. The ability of Bag1L to enhance AR activity was reduced with the removal of an NH(2)-terminal domain of Bag1L, which was found to be required for efficient nuclear localization and/or retention. In contrast, deletion of a conserved ubiquitin-like domain from Bag1L did not interfere with its nuclear targeting or AR regulatory activity. Thus, both the unique NH(2)-terminal domain and the COOH-terminal Hsc70-binding domain of Bag1L are simultaneously required for its function as an AR regulator, whereas the conserved ubiquitin-like domain is expendable.

BAG-family proteins share a conserved protein interaction region, called the 'BAG domain', which binds and regulates Hsp70/Hsc70 molecular chaperones. This family of cochaperones functionally regulates signal transducing proteins and transcription factors important for cell stress responses, apoptosis, proliferation, cell migration and hormone action. Aberrant overexpression of the founding member of this family, BAG1, occurs in human cancers. In this study, a structure-based approach was used to identify interacting residues in a BAG1--Hsc70 complex. An Hsc70-binding fragment of BAG1 was shown by multidimensional NMR methods to consist of an antiparallel three-helix bundle. NMR chemical shift experiments marked surface residues on the second (alpha 2) and third (alpha 3) helices in the BAG domain that are involved in chaperone binding. Structural predictions were confirmed by site-directed mutagenesis of these residues, resulting in loss of binding of BAG1 to Hsc70 in vitro and in cells. Molecular docking of BAG1 to Hsc70 and mutagenesis of Hsc70 marked the molecular surface of the ATPase domain necessary for interaction with BAG1. The results provide a structural basis for understanding the mechanism by which BAG proteins link molecular chaperones and cell signaling pathways.

In the adult small intestine, the dynamic renewal of the epithelium is characterized by a sequence of cell production in the crypts, cell maturation and cell migration to the tip of villi, where apoptosis is undertaken. Little is known about enterocytic apoptosis during development. In man, intestinal architectural features and functions are acquired largely by mid-gestation (18-20 wks); the question whether the establishment of enterocytic apoptotic processes parallels or not the acquisition of other intestinal functional features remains open. In the present study, we approached this question by examining enterocytic apoptosis during development of the human jejunum (9-20 wks gestation), using the ISEL (in situ terminal uridine deoxynucleotidyl nick-end labelling) method. Between 9 and 17 wks, apoptotic enterocytes were not evidenced. However, beginning at the 18 wks stage, ISEL-positive enterocytes were regularly observed at the tip of villi. Since the Bcl-2 family of proteins constitutes a critical checkpoint in apoptosis, acting upstream of the apoptotic machinery, we investigated the expression of six Bcl-2 homologs (Bcl-2, Bcl-X(L), Mcl-1, Bax, Bak, Bad) and one non-homologous associated molecule (Bag-1). By immunofluorescence, we found that all homologs analyzed were expressed by enterocytes between 9 and 20 wks. However, Bcl-2 homologs underwent a gradual compartmentalization of epithelial expression along the maturing crypt-villus axis, to establish gradients of expression by 18-20 wks. Western blot analyses indicated that the expression levels of Bcl-2 homologs were modulated during morphogenesis of the crypt-villus axis, in parallel to their gradual compartmentalization of expression. Altogether, these data suggest that regulatory mechanisms of human enterocytic apoptosis become established by mid-gestation (18-20 wks) and coincide with the maturation of the crypt-villus axis of cell proliferation, differentiation and renewal.

The c-Myc oncoprotein is a transcription factor involved in cellular transformation as well as apoptotic cell death. We show here that over-expression of c-Myc delivered by an adenovirus vector up-regulates endogenous proapoptotic bax mRNA and protein expression in human cells. In contrast, the cytotoxic tumor necrosis factor-related apoptosis-inducing ligand induces cell death without up-regulating bax expression. c-Myc/Max heterodimers bind to canonical E-box elements located in the bax promoter region as demonstrated by electrophoretic mobility shift analysis and DNaseI foot-printing assays. Analysis of bax regulatory region mutants suggests a model involving myc-dependent activation as well as relief of repression through distinct E-box elements. c-Myc-null cells are deficient in bax-promoter activation as compared with wild-type c-Myc-expressing cells. Overexpression of c-Myc in serum-starved human or mouse embryonic cells leads to apoptosis which is significantly reduced in the presence of growth factor-containing serum. c-Myc-induced apoptosis appears to be deficient in bax-null as compared with bax-wild-type mouse embryonic fibroblasts. The results suggest that the cell death-promoting gene bax is directly downstream of c-Myc in a pathway leading to apoptosis.

We investigated the mechanism of lysosome-mediated cell death using purified recombinant pro-apoptotic proteins, and cell-free extracts from the human neuronal progenitor cell line NT2. Potential effectors were either isolated lysosomes or purified lysosomal proteases. Purified lysosomal cathepsins B, H, K, L, S, and X or an extract of mouse lysosomes did not directly activate either recombinant caspase zymogens or caspase zymogens present in an NT2 cytosolic extract to any significant extent. In contrast, a cathepsin L-related protease from the protozoan parasite Trypanosoma cruzi, cruzipain, showed a measurable caspase activation rate. This demonstrated that members of the papain family can directly activate caspases but that mammalian lysosomal members of this family may have been negatively selected for caspase activation to prevent inappropriate induction of apoptosis. Given the lack of evidence for a direct role in caspase activation by lysosomal proteases, we hypothesized that an indirect mode of caspase activation may involve the Bcl-2 family member Bid. In support of this, Bid was cleaved in the presence of lysosomal extracts, at a site six residues downstream from that seen for pathways involving capase 8. Incubation of mitochondria with Bid that had been cleaved by lysosomal extracts resulted in cytochrome c release. Thus, cleavage of Bid may represent a mechanism by which proteases that have leaked from the lysosomes can precipitate cytochrome c release and subsequent caspase activation. This is supported by the finding that cytosolic extracts from mice ablated in the bid gene are impaired in the ability to release cytochrome c in response to lysosome extracts. Together these data suggest that Bid represents a sensor that allows cells to initiate apoptosis in response to widespread adventitious proteolysis.

Growth factors are well known for their participation in the regulation of cell proliferation and survival. However, the intracellular signaling pathways by which growth factors promote survival are still poorly understood. In the present study, using the MIA PaCa-2 cell line, a well-established model of pancreatic cancer cells, we analyzed the roles of ERK1/2 activities in the regulation of cell survival and investigated some of the mechanisms involved.The ability of the MEK inhibitor PD98059 to modulate survival of the MIA PaCa-2 cells was evaluated, and the responses were correlated with expression of Bcl-2 homologs and caspases 1, 3, 6, 8, and 9 activities.Herein, we showed that inhibition of ERK1/2 activities caused (1) a G1 arrest; (2) a down-regulation of the expression levels of the anti-apoptotic homologs Bcl-2, Mcl-1, and Bcl-X(L) without affecting the pro-apoptotic levels of Bax and Bak; (3) a promotion of caspases 3, 6, 8, and 9 activities; (4) a stimulation of PARP cleavage; and (5) a programmed cell death by apoptosis.Our data suggest that activation of the ERK pathway functions to protect pancreatic tumor cells from apoptosis as well as to regulate their progression in the cell cycle.

In this study, we sought to investigate in more detail the role of caspase-3 in apoptotic processes in cultured cells and in cell-free extracts of breast cancer cells. We present evidence that apoptosis of caspase-3-deficient MCF-7 breast cancer cells is defective in response to cisplatin treatment, as determined by chromatin condensation, nuclear fragmentation, DNA fragmentation, and release of cytochrome c from the mitochondria. Reconstitution of MCF-7 cells by stable transfection of CASP-3 cDNA restores all these defects and results in an extensive apoptosis after cisplatin treatment. We further show that in extracts from caspase-3-deficient MCF-7 cells, procaspase-9 processing is strongly impaired after stimulation with either cytochrome c or recombinant caspase-8. Reconstitution of MCF-7 cell extracts with procaspase-3 corrects this defect, resulting in an efficient and complete processing of procaspase-9. Together, our data define caspase-3 as an important integrator of the apoptotic process in MCF-7 breast cancer cells and reveal an essential function of caspase-3 for procaspase-9 processing.

Two methods for assaying Bcl-2 protein family-induced solute efflux from liposomes have been outlined. They utilize either ion-selective electrodes to follow ion efflux or fluorescence to monitor changes in fluorescence of the liposome-encapsulated dye SPQ or carboxyfluorescein. Both methods provide a simple means of determining protein activity. These methods do not have the capability to detect either single-channel conductivity or ion selectivity, but they indicate whether the bulk of the protein population is inducing solute efflux. Although in in vivo significance of Bcl-2 protein family pore formation remains to be determined, in vitro measurements of channel activity should provide a means to determine whether a given protein preparation has activity and whether mutations have an adverse effect on channel formation.

Compounds that inhibit protein kinases are currently undergoing clinical evaluation for the treatment of a variety of malignancies. The kinase inhibitors flavopiridol and 7 hydroxy-staurosporine (UCN-01) were examined for their effects on B-cell chronic lymphocytic leukemia (B-CLL) cells in vitro (n = 49). Flavopiridol and UCN-01 induced concentration-dependent apoptosis of most B-CLL samples tested, with greater than 50% cell killing occurring at concentrations of less than 1 mcmol/L, and with flavopiridol displaying more potent activity than UCN-01. Flavopiridol (0.1 mcmol/L) and UCN-01 (1 mcmol/L) also induced striking decreases in the levels of the antiapoptosis proteins Mcl-1, X-linked inhibitor of apoptosis (XIAP), and BAG-1 in nearly all cases of B-CLL and of Bcl-2 in approximately half of B-CLL specimens evaluated. In contrast, expression of the proapoptotic proteins Bax and Bak was not significantly influenced by these kinase inhibitors. Flavopiridol-induced decreases in the levels of antiapoptosis proteins Mcl-1 and XIAP preceded apoptosis and were not substantially affected by the addition of caspase inhibitors to cultures. In contrast, UCN-01-stimulated decreases in antiapoptosis proteins were slower, occurred concurrently with apoptosis, and were partially prevented by caspase inhibitors. The findings suggest that flavopiridol and UCN-01 induce apoptosis of B-CLL cells through different mechanisms. The potent apoptotic activities of flavopiridol and UCN-01 against cultured B-CLL cells suggest that they may be effective as single agents in the treatment of B-CLL or for sensitizing B-CLL cells to conventional cytotoxic drugs. (Blood. 2000;96:393-397)

Several studies demonstrate that the BCL-2 and BCL-XL antiapoptotic genes are variably expressed in plasma cells of patients with multiple myeloma (MM). However, the plasma cell expression of BAX protein, their major proapoptotic partner, has not been investigated. Our initial Western blot analysis of myeloma cell extracts also suggested patient variability in the expression of BAX, which was not altered by exposure to interleukin 6. To further investigate the significance of BAX expression, we performed immunohistochemistry on archival bone marrow biopsies and compared BAX staining to BCL-2 immunostaining. Expression was first evaluated in 104 patients with reactive plasmacytosis, monoclonal gammopathy of undetermined significance/smoldering MM, or active MM. An increase (P0.05) in expression of both BAX and BCL-2 was detected in MM patients compared with patients with reactive plasmacytosis. Patients with monoclonal gammopathy of undetermined significance/smoldering MM had intermediate values. For correlations with outcome, expression was assessed in 43 patients at diagnosis who were treated with melphalan and prednisone; 30 at diagnosis who were treated with vincristine, Adriamycin, and dexamethasone; and 29 at relapse who were treated with second-line therapy. There was no correlation between BAX or BCL-2 expression and response to chemotherapy or duration of response or between BCL-2 expression and survival. However, patients who demonstrated extremely low plasma cell BAX expression had significantly increased survival. This was true for patients initially treated with melphalan and prednisone or vincristine, Adriamycin, and dexamethasone, as well as patients studied at relapse. BAX expression did not correlate with expression of proliferating cell nuclear antigen used as a marker of proliferation. These data indicate a myeloma-specific increase in BAX expression in plasma cells and suggest that low BAX expression identifies a cohort of patients with long survival, which is not specifically associated with low proliferating cell nuclear antigen expression.

Ectopic expression of Bcl-2 in photoreceptors of mice with retinal degenerative disease slows progression of the disease. BAG-1 has previously been shown to augment the inhibitory effect of Bcl-2 on programmed cell death in cultured cell systems. This study was designed to determine whether the coexpression of BAG-1 and Bcl-2 in the photoreceptors of mice with an autosomal dominant form of retinitis pigmentosa (RP) would enhance the protective effect provided by Bcl-2 alone.An expression vector using the 5' regulatory region of the murine opsin gene was used to target the expression of BAG-1 specifically to photoreceptor cells of mice. The BAG-1 transgenic mice were crossed to Bcl-2 transgenics to obtain animals that coexpress the two transgenes in photoreceptor cells. BAG-1/Bcl-2 animals were then crossed to an RP mouse model (a transgenic line overexpressing the S334ter rhodopsin mutant) to assess the effect of coexpression of BAG-1 and Bcl-2 on retinal degeneration. Morphologic analysis was performed on retinas isolated at various times after birth to monitor disease progression.High levels of BAG-1 expression resulted in retinal degeneration that was not prevented by Bcl-2 expression. However, coexpression of appropriate levels of BAG-1 and Bcl-2 was found to have a profound inhibitory effect on retinal degeneration caused by overexpression of a mutant rhodopsin transgene. Whereas expression of Bcl-2 alone was previously found to delay degeneration of the retina from 2 weeks to approximately 4 weeks of age, coexpression of BAG-1 and Bcl-2 inhibited photoreceptor cell death for as long as 7 to 9 weeks.The synergistic effect against photoreceptor cell death produced by the coexpression of Bcl-2 and BAG-1 indicates that these proteins can function in concert to prevent cell death. At the correct dosage, coexpression of Bcl-2 and BAG-1 may serve as a potential means to treat retinal degenerative diseases.

Expression of several inhibitor of apoptosis proteins (IAPs) was investigated in the National Cancer Institute panel of 60 human tumor cell lines, and the expression and prognostic significance of one of these, XIAP, was evaluated in 78 previously untreated patients with acute myelogenous leukemia (AML). XIAP and cIAP1 were expressed in most cancer lines analyzed, with substantial variability in their relative levels. In contrast, NAIP mRNA was not detectable, and cIAP2 was found at the mRNA and protein levels in only 34 (56%) and 5 (8%) of the 60 tumor cell lines analyzed, respectively. Interestingly, XIAP, cIAP1, and cIAP2 mRNA levels did not correlate with protein levels in the tumor lines, indicating posttranscriptional regulation of expression. High levels of XIAP protein in tumor cell lines were unexpectedly correlated with sensitivity to some anticancer drugs, particularly cytarabine and other nucleosides, whereas higher levels of cIAP1 protein levels were associated with resistance to several anticancer drugs. The relevance of XIAP to in vivo responses to cytarabine was explored in AML, making correlations with patient outcome (n = 78). Patients with lower levels of XIAP protein had significantly longer survival (median, 133 versus 52.5 weeks; P = 0.05) and a tendency toward longer remission duration (median, 87 versus 52.5 weeks; P = 0.13) than those with higher levels of XIAP. Altogether, these findings show that IAPs are widely but differentially expressed in human cancers and leukemias and suggest that higher XIAP protein levels may have adverse prognostic significance for patients with AML.

Caspases are cysteine proteases that are specific for aspastic acid residues. These enzymes have been extensively characterized as integral and highly conserved components of a variety of cell death programs. Cowpox and several insect viruses have evolved mechanisms that counter host cell suicide by encoding proteins that directly inhibit caspases-thereby allowing propagation of viral progeny within the host cell. It has only recently been elucidated, however, that endogenous cellular inhibitors of the caspases exist. To date five members of the inhibitor of apoptosis (IAP) family of proteins has been identified in humans and at least three of these have been shown directly to inhibit specific caspases. Thus, members of the IAP family of proteins are the only endogenous inhibitors of caspases known in mammals. Here we discuss the caspase and IAP families of proteins and review the data concerning their relationship.

CD40 plays a critical role in immunoglobulin (Ig) class switching in B cells, but the molecular events involved remain poorly understood. Using CD40 mutants with impairments in their ability to bind selected TNF receptor-associated factor (TRAF) family proteins, we observed that CD40-mediated transcriptional induction of the germ-line Ig-Cgamma1- and Ig-Cepsilon promoters was markedly reduced by mutations that prevent TRAF2, TRAF3, TRAF5 or TRAF6 binding. Moreover, co-expression of trans-dominant inhibitory forms of TRAF2, 3, 5 or 6 with wild-type CD40 also suppressed induction of these promoters. Overexpression of TRAF2 or TRAF6 was sufficient to induce transcription of the C(H) promoters through an NF-kappaB-dependent mechanism. In contrast, TRAF3 and TRAF5 failed to induce these promoters, implying a more indirect role for these TRAF family members. Altogether, the results demonstrate a non-redundant role for multiple TRAF in the signal transduction pathways by which CD40 induces transcription of germ-line C(H) promoters. Since C(H) germ-line transcription represents an obligatory step in Ig class switching in B cells, these findings suggest that interference with the functions of any of these TRAF might provide a means of preventing class switching for therapeutic purposes.

The present studies compared caspase activation under cell-free conditions in vitro and in etoposide-treated HL-60 leukemia cells in situ. Immunoblotting revealed that incubation of HL-60 cytosol at 30 degrees C in the presence of cytochrome c and ATP (or dATP) resulted in activation of procaspases-3, -6, and -7 but not -2 and -8. Although similar selectivity was observed in intact cells, affinity labeling revealed that the active caspase species generated in vitro and in situ differed in charge and abundance. ATP and dATP levels in intact HL-60 cells were higher than required for caspase activation in vitro and did not change before caspase activation in situ. Replacement of ATP with the poorly hydrolyzable analogs 5'-adenylyl methylenediphosphate, 5'-adenylyl imidodiphosphate, or 5'-adenylyl-O-(3-thiotriphos-phate) slowed caspase activation in vitro, suggesting that ATP hydrolysis is required. Caspase activation in vitro was insensitive to phosphatase and kinase inhibitors (okadaic acid, staurosporine, and genistein) but was inhibited by Zn(2+), aurintricarboxylic acid, and various protease inhibitors, including 3,4-dichloroisocoumarin, N(alpha)-p-tosyl-L-phenylalanine chloromethyl ketone, N(alpha)-p-tosyl-L-lysine chloromethyl ketone, and N-(N(alpha)-benzyloxycarbonylphenylalanyl)alanine fluoromethyl ketone, each of which inhibited recombinant caspases-3, -6, -7, and -9. Experiments with anti-neoepitope antiserum confirmed that these agents inhibited caspase-9 activation. Collectively, these results suggest that caspase-9 activation requires nucleotide hydrolysis and is inhibited by agents previously thought to affect apoptosis by other means.