| Lymphocyte Cell Biology Unit |
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Dan L. Longo, M.D., Scientific Director, NIA
Senior Investigator |
| Overview |
| The Function of Bcl-2: Bcl-2 is known to prevent apoptosis in response to a variety of stimuli. It is said to work through binding to proapoptotic Bcl-2 family members such as Bax and Bad. However, its precise mechanism of action is poorly defined. We have analyzed Bcl-2 function in the prevention of apoptosis from chemotherapeutic agents and other stimuli. We have found that Bcl-2 function can be altered by phosphorylation. Agents that interfere with microtubule function initiate a signalling cascade that among other things, activates jun N-terminal kinase or JNK, which appears to be involved in Bcl-2 phosphorylation. The site on Bcl-2 that is phosphorylated with attendant inhibition of Bcl-2 function is serine 70. A Bcl-2 mutant that lacks serine 70 or its adjacent loop region is hyperfunctional and cannot be inhibited by phosphorylation. We have also found that apoptosis from paclitaxel and vincristine is mediated substantially through the fas/fas ligand pathway. Bcl-2 interferes with paclitaxel-induced apoptosis by blocking the upregulation of fas ligand expression in the tumor cells. It does this by binding to calcineurin and preventing its activation. Thus, the transcription factor, NFAT, remains phosphorylated and nonfunctional and is unable to translocate to the nucleus to induce fas ligand expression. Bcl-2 binds to calcineurin through its BH4 domain. Another unexpected finding in this work was that Bcl-2 appears to be capable of preventing the generation of nitric oxide, an important early step in some forms of apoptosis. Future studies are aimed at understanding the mechanism of Bcl-2 inhibition of nitric oxide generation. |
| The Regulation of Growth Fraction in Tumor Cells: The vast majority of solid tumors have a very low growth fraction at the time they become clinically evident, usually in the range of 3-7%. When the tumor is treated, the growth fraction increases in an effort to maintain the tumor cell mass. This is reminiscent of the organization of most organ systems. Resting bone marrow stem cells are recruited into cycle when under the influence of a myelotoxic stimulus. Surgical removal of a portion of the liver stimulates the recruitment of hepatocytes into the cell cycle to replace the removed tissue. Other examples could also be cited. What is of interest to us is how a tumor cell, with its many genetic abnormalities that tend to promote proliferation, is pulled out of the cell cycle in the first place. Some gene product that is working in the resting tumor cells has managed to antagonize all the oncogene mutations and missing or malfunctioning tumor suppressor gene products and stop the cell from dividing; and it does this reversibly. When the tumor perceives an attack that reduces its volume, cells can be recruited back into the cell cycle. We are separating fresh lymphoma specimens into dividing and nondividing populations, isolating cDNA, and using differential display and microarray techniques, characterizing genes that are expressed in resting cells but not in dividing cells. Such messages will be isolated, their genes identified, and then the message will be introduced into dividing cells to look for growth arrest. |
| Tumor-induced Immunosuppression: We initially observed, and it has been widely reproduced, that T cells from tumor-bearing hosts are defective in their signalling in response to antigen and in their function. A variety of defects are noted including defective nuclear translocation of the p65 NF-kappa B transcription factor, shortened half-lives for a number of cellular proteins such as TCR-zeta chain and signalling kinases of the src family, among others, and a deviation of the cytokine production profile toward Th2 cytokines (IL-4, IL-10) and away from Th1 cytokines (interferon-gamma, TNF). Evidence of suppression of immune function in mice in which tumor is growing in hollow fibers in the peritoneal cavity without any cell-cell contact in the host suggests that a soluble tumor factor is responsible for the defect in cellular immunity. We have devised a method of reproducing these tumor-induced changes in normal NK cells in vitro and are in the process of isolating the tumor-derived factor responsible for the changes. |
