Nuclear partners of Bcl-2: Bax and PML.

A milestone in understanding the functioning of the antiapoptotic cytoplasmic protein Bcl-2 was the discovery that Bcl-2 was capable of heterodimerising with the pro-apoptotic protein Bax at the mitochondrial level, creating a delicate balance of cell death preventing and promoting regulators. In recent years we identified substantial pools of Bcl-2 and Bax in nucleoplasm as well. We demonstrated that nuclear Bcl-2 controls cellular proliferation and, in an indirect manner, apoptosis. Sound support for functional presence of nuclear Bcl-2 and Bax would be evidence of Bcl-2-Bax binding in this compartment. Here we show by immunoprecipitation-using a battery of commercially available, monoclonal antibodies-that Bcl-2 binds Bax in nuclei of human breast cancer cells. Interestingly, findings by others pointed at an interaction between the product of the promyelocytic leukemia gene, the PML protein, and Bax. PML plays a part in cell proliferation and apoptosis, a rather similar role we assigned to nuclear Bcl-2. Nuclear Bcl-2, but not Bax, was found to immunoprecipitate with nuclear PML. These data show that binding of Bcl-2 with structurally and functionally related proteins extends to the nucleus, emphasizing its pivotal role in Bcl-2-mediated actions.

The role of various Bcl-2 domains in the anti-proliferative effect and modulation of cellular glutathione levels: a prominent role for the BH4 domain.

Reduced cell proliferation and increased levels of cellular glutathione (GSH) are characteristic for cells that overexpress the anti-apoptotic Bcl-2 protein. We investigated the influence of various Bcl-2 domains on both these characteristics. Rat CC531 colorectal cancer cells were stably transfected with the human bcl-2 gene (CCbcl2 cells) or with bcl-2 gene constructs missing a coding sequence for a func-tional domain, BH1 (CCDeltaBH1 cells), BH3 (CCDeltaBH3 cells), BH4 (CCDeltaBH4 cells) or the transmembrane region (CCDeltaTM cells). We measured GSH levels in exponentially and confluent growing bcl-2-transfected cell populations. The fraction of S-phase cells during exponential growth was significantly reduced in CCbcl2, CCDeltaBH1, CCDeltaBH3, and CCDeltaTM cells compared with parental CC531, neo-transfected CC531 and CCDeltaBH4 cells. GSH levels in these bcl-2 transfectants were significantly higher than in the parental line measured at 50% confluence; at 100% confluence they reached a similar level as found in parental cells. Independently from the presence of BH1, BH3 or TM domains, overexpression of Bcl-2 reduces cellular proliferation under conditions of increased GSH levels. This apparent link is lost in CCDeltaBH4 cells; these cells are not reduced in cellular proliferation and harbour significantly higher GSH levels than found in the other transfectants. Studies on the subcellular localization revealed an extremely low expression of the Bcl-2 protein lacking the N-terminal BH4 domain in nuclear fractions. Nuclear translocation of Bcl-2 requires the presence of the BH4 domain and seems prominent in reducing cellular proliferation.

The presence of 19-kDa Bcl-2 in dividing cells.

The 26-kDa bcl-2 gene product inhibits apoptosis and cell proliferation. Cleavage of Bcl-2 into a 22-kDa fragment inactivates its anti-apoptotic activity and is a key event in apoptosis. Here, and in recent work, we describe massive 19-kDa Bcl-2 immunoreactivity in non-apoptotic cells, suggesting a link with viability rather than cell death. Loss of 19 kDa Bcl-2 in adriamycin-induced apoptotic cells underlines this. G2/M-phase accumulation of cells by nocodazole-treatment also results in loss of 19 kDa Bcl-2. Next to its well-documented cytoplasmic localization, a substantial pool of Bcl-2 resides in nuclei. Hampered nuclear localization of Bcl-2 leads to a loss of cell cycle repression. This has led us to point at a pivotal role for nuclear Bcl-2 in cellular proliferation. In this report, cellular fractionation of bcl-2 transfected cells in various phases of the cell cycle reveals a constitutive cytoplasmic pool of 19 kDa Bcl-2. Nuclear 19-kDa Bcl-2 immunoreactivity is far more pronounced in rapidly dividing nuclei compared with more quiescent nuclear fractions. This implicates that ongoing cell proliferation involves cleavage of nuclear Bcl-2 with a 19-kDa fragment.

Comparison of the effects of microwave heating and high pressure cooking for antigen retrieval of human and rat Bc1-2 protein in formaldehyde-fixed, paraffin-embedded sections.

Immunohistochemical detection of expression of the anti-apoptotic Bcl-2 protein is widely studied as a putative prognostic and predictive factor in various types of cancer. For that purpose, heating for 10 min by microwave (MW) up t o 100 degrees C in citrate buffer, pH 6.0, prior to immunostaining is often used to retrieve Bcl-2 antigens in archival formalin-fixed, paraffin-embedded tissue. We recently reported that Bcl-2 is not only a cytoplasmic protein, but that it is present also in interphase nuclei and that it strongly associates with mitotic chromosomes. Furthermore, we showed that binding of the monoclonal antibody (MAb) #124 with nuclear/chromosomal epitopes is diminished by formaldehyde-based fixatives and cannot be restored by MW treatment for 10 min. Here we report that prolonged MW heating or heating up to 130 degrees C in a high pressure cooker (HPC), despite improved cytoplasmic immunostaining, fails to retrieve nuclear/chromosomal Bcl-2 epitopes recognized by the MAb #124 in human tissues. In contrast, these procedures can retrieve nuclear/chromosomal Bcl-2 epitopes detected by polyclonal #15616E antibodies in rat tissues. The specificity of these epitopes was confirmed by Western blot analysis of tissues treated by MW heating or HPC.

Routine formaldehyde fixation irreversibly reduces immunoreactivity of Bcl-2 in the nuclear compartment of breast cancer cells, but not in the cytoplasm.

Bcl-2 and Bax belong to a family of proteins involved in apoptosis regulation and are believed to reside in the cellular cytoplasm. The authors recently reported interphase nuclear localization of both proteins after immunofluorescence staining of formaldehyde- and methanol-fixed human and rodent cell monolayers. In addition, the authors' data confirmed earlier reports on Bcl-2 immunoreactivity of mitotic chromosomes in human cells. In their experience, nuclear or mitotic staining of Bcl-2, in contrast with cytoplasmic Bcl-2 immunoreactivity, is rarely observed in formaldehyde-fixed, paraffin-embedded breast cancer specimens. Therefore, the authors wondered if nuclear and mitotic Bcl-2 immunoreactivity could be irreversibly reduced by certain fixation procedures, including formaldehyde fixation. Here the authors investigated the effects of various routinely used fixation protocols and antigen retrieval techniques on Bcl-2 and Bax immunoreactivity in monolayers of MCF-7 human breast cancer cells. Whereas nuclear and mitotic immunoreactivity for Bcl-2 was clearly present after formaldehyde and methanol fixation, it was completely absent in cells fixed in acetone, methanol, or formaldehyde alone. In addition, it was found that in particular nuclear and mitotic Bcl-2, and to a lesser extent cytoplasmic Bcl-2 immunoreactivity, decreased after prolonged formaldehyde fixation, whereas Bax immunoreactivity diminished only slightly. Heat-mediated antigen retrieval after prolonged formaldehyde fixation elevated cytoplasmic, but not nuclear and mitotic, Bcl-2 immunoreactivity.

Effects of acetone, methanol, or paraformaldehyde on cellular structure, visualized by reflection contrast microscopy and transmission and scanning electron microscopy.

The authors recently showed variable subcellular immunoreactivity of the Bcl-2 and Bax proteins after fixation of cell monolayers with acetone, methanol, or paraformaldehyde (PF) followed by methanol (PF/methanol). Here, the authors demonstrate by reflection contrast microscopy and transmission electron microscopy that acetone or methanol fixation result in complete loss of integrity of intracellular structures in contrast with PF or glutaraldehyde fixation. Scanning electron microscopy revealed poor preservation of plasma membrane integrity after fixation in acetone or methanol. Fixation with PF before methanol reduced damage to intracellular and plasma membranes. In addition, Western blot analysis demonstrated loss of Bcl-2 and Bax protein during acetone or methanol fixation, whereas PF fixation before methanol permeabilization markedly reduced this loss. For studies on the intracellular localization of soluble or unknown types of antigen, the authors discourage the use of acetone and methanol as single fixatives.

Decreased exposure to saquinavir in HIV-1-infected patients after long-term antiretroviral therapy including ritonavir and saquinavir.

OBJECTIVE: To explore whether steady-state plasma pharmacokinetics of ritonavir and saquinavir change during long-term treatment in HIV-1-infected patients on antiretroviral treatment including ritonavir and saquinavir. METHODS: The pharmacokinetics of ritonavir and saquinavir were assessed during an 8-h period on two occasions in six HIV-1 infected patients on stable twice daily treatment with ritonavir 400 mg, saquinavir 400 mg and stavudine 40 mg with or without lamivudine 150 mg twice daily. RESULTS: The first study day was 4-12 months (median 7 months) after the start of the current regimen. The second study day was 9-15 months (median 10 months) later. No significant differences were observed for the ritonavir pharmacokinetics between the first and second study day. However, median change in plasma trough level of saquinavir between the two study days was -30% (range -79 to +11%; P = 0.06). Median change in maximum plasma concentration was -40% (range -62 to +34%; P = 0.09). The median change in area under the plasma concentration versus time curve over 0-8 h was -33% (range -53 to +21%; P = 0.06). CONCLUSION: The exposure to saquinavir decreased over time in HIV-infected patients on stable antiretroviral therapy. These data suggest that regular monitoring of plasma drug concentrations should become part of routine patient care even in apparently compliant patients.

Development of resistance to glutathione depletion-induced cell death in CC531 colon carcinoma cells: association with increased expression of bcl-2.

The glutathione (GSH) level of CC531 rat colorectal cancer cells is readily decreased by exposure to buthionine sulfoximine (BSO), an inhibitor of GSH synthesis; at 25 microM BSO, these cells died in a non-apoptotic fashion. By continuous exposure of CC531 cells to increasing concentrations of BSO, we obtained a BSO-resistant cell line (CCBR25) that was 50 times more resistant to BSO than the parental cell line. Whereas the GSH content of CCBR25 and CC531 cells was similar, the former contained a much higher level of the Bcl-2 protein. After stable transfection of CC531 cells with the human bcl-2 gene, the resulting Bcl-2-overexpressing cell line appeared to be 9 times more resistant to BSO than the parental cell line. These findings suggest that the Bcl-2 protein offers resistance against the cytotoxic effect of severe GSH depletion.

Corticosterone regulates expression of BDNF and trkB but not NT-3 and trkC mRNA in the rat hippocampus.

Corticosterone has profound effects on growth, differentiation, and synaptic transmission of hippocampal neurons by activation of mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs). In the present study we tested if neurotrophins can be implicated in these effects. For this purpose we injected 30, 300, and 1,000 microg corticosterone s.c. (per kg body weight) in adrenalectomized rats and measured the mRNA levels of brain-derived neurotrophic factor (BDNF), tyrosine receptor kinase (trk)B, neurotrophin (NT)-3, and trkC in hippocampal cell fields at 6 hr after steroid administration by in situ hybridization. NT-3 and trkC mRNA did not show significant changes in any hippocampal region after the various doses of corticosterone. BDNF mRNA decreased after corticosterone administration dose dependently, resulting in a maximal suppression of 35, 20, and 50% in dentate gyrus, CA3, and CA1, respectively. Interestingly, trkB responded to corticosterone in an inverted U-shaped fashion in CA3 and dentate gyrus: the low dose of corticosterone increased trkB mRNA expression in both regions by approximately 30%, while the effect of the two higher doses was not different from the vehicle injected controls. In conclusion, we found differential effects of low and high doses of corticosterone on BDNF and trkB expression in hippocampus, which suggests involvement of a coordinated MR- and GR-mediated action.