In multicellular organisms, homeostasis is maintained through a balance between cell proliferation and cell death. Although much is known about the control of cell proliferation, less is known about the control of cell death. Physiologic cell death occurs primarily through an evolutionarily conserved form of cell suicide termed apoptosis. The decision of a cell to undergo apoptosis can be influenced by a wide variety of regulatory stimuli. Recent evidence suggests that alterations in cell survival contribute to the pathogenesis of a number of human diseases, including cancer, viral infections, autoimmune diseases, neurodegenerative disorders, and AIDS (acquired immunodeficiency syndrome). Treatments designed to specifically alter the apoptotic threshold may have the potential to change the natural progression of some of these diseases.

https://science.sciencemag.org/content/sci/267/5203/1456.full.pdf

Apoptosis in the pathogenesis and treatment of disease

Apoptosis is a distinct mode of cell death that is responsible for deletion of cells in normal tissues; it also occurs in specific pathologic contexts. Morphologically, it involves rapid condensation and budding of the cell, with the formation of membrane-enclosed apoptotic bodies containing well-preserved organelles, which are phagocytosed and digested by nearby resident cells. There is no associated inflammation. A characteristic biochemical feature of the process is double-strand cleavage of nuclear DNA at the linker regions between nucleosomes leading to the production of oligonucleosomal fragments. In many, although not all of the circumstances in which apoptosis occurs, it is suppressed by inhibitors of messenger RNA and protein synthesis. Apoptosis occurs spontaneously in malignant tumors, often markedly retarding their growth, and it is increased in tumors responding to irradiation, cytotoxic chemotherapy, heating and hormone ablation. However, much of the current interest in the process stems from the discovery that it can be regulated by certain proto-oncogenes and the p53 tumor suppressor gene. Thus, c-myc expression has been shown to be involved in the initiation of apoptosis in some situations, and bcl-2 has emerged as a new type of proto-oncogene that inhibits apoptosis, rather than stimulating mitosis. In p53-negative tumor-derived cell lines transfected with wild-type p53, induction of the gene has, in rare cases, been found to cause extensive apoptosis, instead of growth arrest. Finally, the demonstration that antibodies against a cell-surface protein designated APO-1 or Fas can enhance apoptosis in some human lymphoid cell lines may have therapeutic implications.

Apoptosis. Its significance in cancer and cancer Therapy

Apoptosis is a morphologically and biochemically distinct form of cell death that occurs under a variety of physiological and pathological conditions. In the present study, the proteolytic cleavage of poly(ADP-ribose) polymerase (pADPRp) during the course of chemotherapy-induced apoptosis was examined. Treatment of HL-60 human leukemia cells with the topoisomerase II-directed anticancer agent etoposide resulted in morphological changes characteristic of apoptosis. Endonucleolytic degradation of DNA to generate nucleosomal fragments occurred simultaneously. Western blotting with epitope-specific monoclonal and polyclonal antibodies revealed that these characteristic apoptotic changes were accompanied by early, quantitative cleavage of the M(r) 116,000 pADPRp polypeptide to an M(r) approximately 25,000 fragment containing the amino-terminal DNA-binding domain of pADPRp and an M(r) approximately 85,000 fragment containing the automodification and catalytic domains. Activity blotting revealed that the M(r) approximately 85,000 fragment retained basal pADPRp activity but was not activated by exogenous nicked DNA. Similar cleavage of pADPRp was observed after exposure of HL-60 cells to a variety of chemotherapeutic agents including cis-diaminedichloroplatinum(II), colcemid, 1-beta-D-arabinofuranosylcytosine, and methotrexate; to gamma-irradiation; or to the protein synthesis inhibitors puromycin or cycloheximide. Similar changes were observed in MDA-MB-468 human breast cancer cells treated with trifluorothymidine or 5-fluoro-2'-deoxyuridine and in gamma-irradiated or glucocorticoid-treated rat thymocytes undergoing apoptosis. Treatment with several compounds (tosyl-L-lysine chloromethyl ketone, tosyl-L-phenylalanine chloromethyl ketone, N-ethylmaleimide, iodoacetamide) prevented both the proteolytic cleavage of pADPRp and the internucleosomal fragmentation of DNA. The results suggest that proteolytic cleavage of pADPRp, in addition to being an early marker of chemotherapy-induced apoptosis, might reflect more widespread proteolysis that is a critical biochemical event early during the process of physiological cell death.

https://cancerres.aacrjournals.org/content/canres/53/17/3976.full.pdf

Specific Proteolytic Cleavage of Poly(ADP-ribose) Polymerase: An Early Marker of Chemotherapy-induced Apoptosis

Programmed cell death (PCD) or apoptosis is a naturally occurring cell suicide pathway induced in a variety of cell types. In many cases, PCD is induced by the withdrawal of specific hormones or growth factors that function as survival factors. In this study, we have investigated the potential role of the extracellular matrix (ECM) as a cell survival factor. Our results indicate that in the absence of any ECM interactions, human endothelial cells rapidly undergo PCD, as determined by cell morphology, nuclei fragmentation, DNA degradation, protein cross-linking, and the expression of the PCD-specific gene TRPM-2. PCD was blocked by plating cells on an immobilized integrin beta 1 antibody but not by antibodies to either the class I histocompatibility antigen (HLA) or vascular cell adhesion molecule-1 (VCAM-1), suggesting that integrin-mediated signals were required for maintaining cell viability. Treatment of the cells in suspension with the tyrosine phosphatase inhibitor sodium orthovanadate also blocked PCD. When other cell types were examined, some, but not all, underwent rapid cell death when deprived of adhesion to the ECM. These results suggest that in addition to regulating cell growth and differentiation, the ECM also functions as a survival factor for many cell types.

The extracellular matrix as a cell survival factor.

In primates, prefrontal, inferior temporal, and posterior parietal cortex are important for cognitive function. It is shown that in adult macaques, new neurons are added to these three neocortical association areas, but not to a primary sensory area (striate cortex). The new neurons appeared to originate in the subventricular zone and to migrate through the white matter to the neocortex, where they extended axons. These new neurons, which are continually added in adulthood, may play a role in the functions of association neocortex.

/pdf/neurogenesis-in-the-neocortex-of-adult-primates-13ac694cy1.pdf

Neurogenesis in the Neocortex of Adult Primates

RNA and protein products encoded by the testosterone-repressed prostate message-2 gene (TRPM-2) are induced to high levels, coordinate with the onset of cell death, in numerous rodent models of inducible tissue damage. These models include cell death initiated by hormonal stimuli (prostate regression), pressure insult (renal atrophy after ureteral obstruction), developmental stimuli (necrosis of interdigital tissue), and cytotoxic injury (chemotherapeutic regression of a tumor). Sequence analysis of cDNA encoding TRPM-2 revealed its close homology with a product referred to as SGP-2 or clusterin expressed constitutively by Sertoli cells; however, the immunologically related polypeptides expressed in regressing tissues differ in molecular mass from the forms secreted by the testis. Although the function(s) of the products encoded by the TRPM-2 gene remains unclear, their presence provides a remarkable and early indicator of programmed cell death in many types of mammalian cells.

Induction of the TRPM-2 gene in cells undergoing programmed death.

Castration initiates extensive apoptosis of the secretory epithelial cells lining the ducts of the rat ventral prostate, resulting in the striking regression of this male sexual accessory tissue. We had previously described the paradox of finding similar cascades of gene activity (c-fos greater than c-myc greater than hsp-70) induced during the early period of ventral prostate regression and during the regrowth of the ventral prostate gland initiated by testosterone replenishment. This common pattern of protooncogene expression during periods of predominant cellular apoptosis or proliferation caused us to examine further the possibility that the two cellular events occur through identical early molecular pathways. In the present study we demonstrate that apoptotic prostate epithelial cells incorporate bromodeoxyuridine into nuclear high-molecular-weight DNA prior to nuclear DNA fragmentation. The DNA synthetic activity occurs in coordination with a massive induction of proliferative cell nuclear antigen, a proliferation marker, in the nuclei of androgen-deprived prostatic epithelial cells. Moreover, this activity is also associated with the increased expression of mRNA encoding p53, a suppressor gene well known as a cell cycle-blocking agent. Our data indicate that quiescent (G0) prostate epithelial cells undergo apoptosis due to two sequential events initiated by testosterone depletion. The first event is the active reentry of these cells into the cell cycle. The second event is the apoptotic destruction resulting from the inability of the differentiated cells to successfully complete this cycle.

https://cancerres.aacrjournals.org/content/canres/52/16/4313.full.pdf

Hormone-regulated apoptosis results from reentry of differentiated prostate cells onto a defective cell cycle.

BACKGROUND

Recent hypotheses to explain the etiology of abnormal growth associated with prostate disease have invoked perturbations in the rate of apoptosis as an important contributor to the onset and progression of these diseases. For this reason, the apoptosis suppressing oncoprotein bcl-2 has come under scrutiny with regards to its role in prostate diseases. In order to evaluate the role of bcl-2 in human prostate disease and to develop an animal model to test anti-bcl-2 therapies, we generated transgenic mice in which bcl-2 expression is targeted to the mouse prostate gland.



METHODS

Mouse embryos were microinjected with recombinant DNA constructed by fusing a modified rat C3(1) promotor element to cDNA encoding human bcl-2. Presence of the C3(1)-bcl-2 transgene in progeny was identified by Southern blot and polymerase chain reaction (PCR) analysis. RNase protection assays were used to analyze RNA from 15 organs of these mice. Western blot assays and immunohistochemical staining were used to confirm the tissue-specific protein expression of human bcl-2 and its cellular localization.



RESULTS

Three lines of C3(1)-bcl-2 transgenic mice were established. Founder mice carried 2–20 copies of the transgene. Expression of human bcl-2 from the transgene was limited to the prostate gland and testis of males as well as the uterus of females. In the prostate gland, human bcl-2 protein was found only in prostatic epithelial cells. Microscopic analysis of prostate glands from individual males (three lines) showed that these glands were often abnormal, with increased accumulation of cells in the prostatic stroma as well as the epithelium.



CONCLUSIONS

These transgenic mice appear to provide a novel animal model for studying neoplastic development of the prostate, with particular emphasis on the bcl-2 protein and the role of apoptosis regulation in such development. Prostate 32:16–26, 1997. © 1997 Wiley-Liss, Inc.

Po-Ying Ng

Papers

Induction of the TRPM-2 gene in cells undergoing programmed death.

Hormone-regulated apoptosis results from reentry of differentiated prostate cells onto a defective cell cycle.

Abnormal prostate development in C3(1)-bcl-2 transgenic mice.

Hormonal Control of Gene Expression in Rat Ventral Prostate: Effects of Androgen Withdrawal on the Expression of Growth-Related Genes