Pergamon Leukemra Research Vol. 20, No. 2, 109-111, 1996. pp.

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COMMENTARY

A DAY IN THE LIFE OF THE Bcl-2 PROTEIN: DOES THE TURNOVER RATE OF Bcl-2 SERVE AS A BIOLOGICAL CLOCK FOR CELLULAR

LIFESPAN REGULATION?

John C. Reed La Jolla Cancer Research Foundation, 10901 N. Torrey Pines Rd, La Jolla, CA 92037, U.S.A.

Since its first discovery 10 years ago at the breakpoints of t(14;18) chromosomes in follicular non-Hodgkin’s lymphomas [l], the BCL-2 gene has assumed a center stage role in leukemia and lymphoma research. The BCL-2 gene provided cancer researchers with the first example of a human proto-oncogene that contributes to the expansion of neoplastic cells by preventing cell death, rather than accelerating cell proliferation [2]. In the hematopoietic and lymphoid systems, billions of new cells are produced daily, necessitating that eradica- tion of cells occurs at a commensurate rate if overall cell numbers are to be maintained within appropriate ranges. The physiological cell death mechanisms that help to maintain homeostasis in hematolymphoid organs, as well as a wide variety of other tissues, are termed programmed cell death or apoptosis. Apoptosis can be induced by a myriad of circumstances and stimuli ranging from growth factor deprivation and treatment with cytotoxic lymphokines (e.g. TNF, Fas-ligand) to infection by viruses and exposure to radiation or chemotherapeutic drugs. Interestingly, gene transfer- mediated elevations in the Bcl-2 protein have been shown to block or substantially delay cell death induced by nearly all apoptotic insults, implying that Bcl-2 controls a distal step in what appears to be an evolutionarily conserved final common pathway for apoptotic cell death (reviewed in [3]). Of potential relevance to the treatment of patients with leukemia and lymphoma, this anti-cell death effect of Bcl-2 also applies to apoptosis induced by essentially all currently available anti-cancer drugs, suggesting that chemother- apeutic agents also utilize a Bcl-2 regulated cell death pathway to ultimately kill malignant cells (reviewed in [41).

Normally, the in vivo patterns of BCL-2 expression coincide with the lifespans of various types of cells, with Bcl-2 protein levels for example declining in myeloid progenitor cells as they undergo differentiation to mature granulocytes, while increasing in thymocytes as they traverse the thymic selection gauntlet to emerge

as longer-lived mature T lymphocytes [5,6]. Dysregula- tion of BCL-2 expression, leading to elevated levels of BCL-2 mRNAs and protein, has been reported in significant percentages of AMLs, CLLs and ALLs [7- 9]. Often, the relative levels of Bcl-2 protein in leukemic cells correlate with the duration of their survival when placed into in vitro culture, suggesting that leukemic cells with higher levels of Bcl-2 enjoy a selective survival advantage [8,9]. Moreover, antisense-mediated reductions in Bcl-2 protein levels have been shown to accelerate the rate of death in cultures of pre-B cell leukemia, T-ALL, non-Hodgkin’s B cell lymphoma and AML cells, as well as to render leukemia and lymphoma cells more sensitive to the cytotoxic effects of anti- cancer drugs in vitro [lO-141. Higher percentages of Bcl-2 positive blasts also have been associated with poor responses to chemotherapy and shorter survival in patients with AML [7]. Taken together, therefore, these observations suggest an important role of dysregulated BCL-2 expression in the pathogenesis and prognosis of at least some types of leukemia and lymphoma.

For the most part, the mechanisms responsible for this dysregulation of BCL-2 gene expression in leukemic cells remain unknown but typically do not involve gross structural alterations to the BCL-2 gene, unlike the situation with approximately two-thirds of non-Hodg- kin’s B-cell lymphomas where t(14:18) translocations place the BCL-2 gene from chromosome 18 under the control of enhancer elements associated with the immunoglobulin (Ig) heavy-chain locus on chromosome 14. What, then, are the molecular events that account for the high levels of Bcl-2 protein found in some leukemias? In this issue, Blagosklonny and colleagues describe the first example of differential regulation of Bcl-2 protein levels due to variations in protein half-life [15]. Specifically, the half-life of Bcl-2 protein, as determined by 35S-methionine pulse-chase analysis, was estimated to be 38 h in an adriamycin-resistant subclone of the HL-60 leukemia line (HL-60/ADR) compared to 20 h in the parental cells. Interestingly, when differ-

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110 J. C. Reed

entiation of HL-60/ADR cells was induced with phorbol ester, BCL-2 mRNA levels rapidly declined and Bcl-2 protein synthesis was abrogated within 8 h, but Bcl-2 protein persisted for >4 days. Due to the normally long half-life of the Bcl-2 protein (2 10 h) relative to the BCL-2 mRNA tl12 (~3 h) [16], a lag in the decline in B&2 protein levels relative to mRNA has been observed previously in other scenarios where a decrease in BCL-2 gene expression is induced, including in a murine myeloblastic leukemia line where ~53 inhibits BCL-2 expression [17], HL-60 cells induced to differ- entiate with retinoids [18], B cell lymphoma cells treated with antisense oligonucleotides [12], and a murine B cell lymphoma line treated with anti-Ig [19]. The paper from Neckers’ group, however, provides the first example where the half-life of the Bcl-2 protein is differentially regulated within a cell line and its derivatives, and raises the possibility that one mechan- ism that may contribute to dysregulation of BCL-2 expression in leukemias is prolongation of the Bcl-2 protein half-life.

The findings also have potentially important implica- tions with regards to regulation of the lifespan of terminally differentiated hematopoietic cells. Because BCL-2 mRNA levels fell and Bcl-2 protein synthesis stopped quickly after stimulating HL-60 cells with phorbol esters, whereas Bcl-2 protein persisted for several days, the differential regulation of Bcl-2 protein production and degradation may provide a mechanism for coordinating cell differentiation with cell death. In this way, differentiation-inducing agents could re- program the transcriptional machinery of progenitor cells for terminal differentiation, and simultaneously create a time-limit on the lifespan of these terminally differentiated cells which would be dictated by the rate of degradation of the Bcl-2 protein. One caveat about the paper in this issue, however, is that attempts were not made to correlate the differential regulation of Bcl-2 protein turnover in parental HL-60 and drug-resistant HL-60/ADR cells with length of survival of the cells after differentiation, and thus we do not know whether the slower decay of Bcl-2 protein in HL-60/ADR cells resulted in prolonged survival of the differentiated cells.

At this point, many questions remain unanswered about the differential regulation of Bcl-2 protein turn- over reported here. For example, how widespread is this phenomenon among leukemic and other types of malignant cells and does it contribute directly or indirectly to the emergence of chemoresistant cells such as the HL-60/ADR subclone studied by Blagosklonny et al. [15]? Given that the Bcl-2 protein normally has a relatively long half-life, for instance, does it really make a difference in the biology of leukemic cells if this half- life is extended further? Also unknown are the molecular mechanisms that account for the differential

rates of Bcl-2 protein degradation in HL-60 and HL-601 ADR cells. In this regard, the Bcl-2 protein is known to interact with several other proteins, including homologs such as Bax, Bcl-X and Mel-1, which are members of the Bcl-2 protein family and non-homologous proteins such as BAG-l, Nip-l, Nip-2 and Nip-3 [20-221. It is possible that some of these Bcl-2-binding proteins influence the half-life of Bcl-2. The BAG-l protein, for example, contains a ubiquitin-like domain [21], and the Bcl-2 homolog Mel-1 as well as the Nip-l, Nip-2 and Nip-3 proteins contain PEST sequences [22,23], thus suggesting potential mechanisms for targeting Bcl- 2 for proteolytic degradation through formation of complexes with Bcl-2. Other possible mechanisms that might contribute to differential regulation of Bcl-2 protein half-life in leukemic cells include phosphoryla- tion of the Bcl-2 protein [24] and changes in the regulation or expression of proteases. With further elucidation of the mechanisms responsible for the elevated levels of Bcl-2 protein found in leukemic and other malignant cells, hopefully it will eventually be possible to devize approaches for selectively reducing Bcl-2 protein levels or function in neoplastic cells, thus rendering them more sensitive to the apoptotic actions of currently available chemotherapeutic drugs and radia- tion.

AcknowZedgement-Dr Reed is a Scholar of the Leukemia Society of America.

References

Tsujimoto Y., Cossman J., Jaffe E. & Croce C. (1985) Involvement of the BCL-2 gene in human follicular lymphoma. Science 228, 1440. Vaux D. L., Cory S. & Adams J. M. (1988) BCL-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells. Nature 335, 440. Reed J. C. (1994) Bcl-2 and the regulation of programmed cell death. J. Cell Biol. 124, 1. Reed J. C. (1995) Bcl-2: prevention of apoptosis as a mechanism of drug resistance. Hematol. Oncol. CZin. N. Am. 9, 451.

5. Delia D., Aiello A., Soligo D., Fontanella E., Melani C., Pezzella F., Pierotti M. A. & Porta G. D. (1992) BCL-2 proto-oncogene expression in normal and neoplastic human myeloid cells. Blood 79, 1291.

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7. Campos L., Roualult J.-P., Sabido O., Roubi N., Vasselon C., Archimbaud E., Magaud J.-P. & Guyotat D. (1993) High expression of Bcl-2 protein in acute myeloid leukemia cells is associated with poor response to chemotherapy. Blood 81, 3091.

8. Hanada M., Delia D., Aiello A., Stadtmauer E. & Reed J. C. (1993) BCL-2 gene hypomethylation and high-level expression in B-cell chronic lymphocytic leukemia. BZood 82, 1820.

A day in the life of the Bcl-2 protein 111

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23. Kozopas K. M., Yang T., Buchan H. L., Zhou P. & Craig R. (1993) Mel-1, a gene expressed in programmed myeloid cell differentiation, has sequence similarity to BCL-2. Proc. natn. Acad. Sci. USA 90, 3516.

24. Haldar S., Jena N. & Croce C. M. (1995) Inactivation of BCL-2 by phosphorylation. Proc. natn. Acad. Sci. USA 92, 4507.