Publisher Summary The classification of cell death can be based on morphological or biochemical criteria or on the circumstances of its occurrence. Currently, irreversible structural alteration provides the only unequivocal evidence of death; biochemical indicators of cell death that are universally applicable have to be precisely defined and studies of cell function or of reproductive capacity do not necessarily differentiate between death and dormant states from which recovery may be possible. It has also proved feasible to categorize most if not all dying cells into one or the other of two discrete and distinctive patterns of morphological change, which have, generally, been found to occur under disparate but individually characteristic circumstances. One of these patterns is the swelling proceeding to rupture of plasma and organelle membranes and dissolution of organized structure—termed “coagulative necrosis.” It results from injury by agents, such as toxins and ischemia, affects cells in groups rather than singly, and evokes exudative inflammation when it develops in vivo. The other morphological pattern is characterized by condensation of the cell with maintenance of organelle integrity and the formation of surface protuberances that separate as membrane-bounded globules; in tissues, these are phagocytosed and digested by resident cells, there being no associated inflammation.

Cell death : the significance of apoptosis

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

The developmental types of secretory cavities in Citrus remain unclear and whether or not programmed cell death is involved in the developmental process of secretory cavities remains an enigma. Regarding cavity formation in Citrus sinensis fruits, this work uncovered novel evidence to delineate secretory cavity formation in schizolysigeny, supporting the possibility of utilizing secretory cavities as a new cell biology model for investigating the molecular mechanism of plant programmed cell death.

Apoptosis. The role of the endonuclease

I. Introduction IN 1953 lohn Morris, an obstetrician at Yale University, reported a series of 82 individuals (80 cases collated from the literature and two cases of his own) who had a female phenotype despite the presence of bilateral testes (1). Since his initial description, studies of the endocrinology, pathophysiology, biochemistry, and molecular biology of the androgen insensitivity syndrome (AIS) have provided insights into the role of androgens in male sex differentiation, the mechanisms of androgen action, and aspects of the structure/function relationships of the androgen receptor. AIS is an archetypal example of a hormone resistance disorder. Androgens are secreted by the testes of these 46,XY individuals in normal or increased amounts; however, due to defective androgen receptor (AR1) function, there is loss of target organ response to the hormone, and the effects of androgens are reduced or absent. Clinical disorders of the AR are reported far more commonly than resistance disorders of other m...

Androgen receptor defects : Historical, clinical, and molecular perspectives

With the aid of monoclonal antibodies specific to the estrogen and progestin receptors, we have examined the cellular localization of these proteins in the reproductive tract of male and female macaques. Two striking findings have resulted from our work with these new reagents. First, these receptors are detectable only in cell nuclei, regardless of hormonal treatment, and second, they are often detectable in stromal, but not epithelial cells when the epithelial cells undergo various estrogen or progestin-dependent events. The latter observation has led us to conclude that stromal cell-epithelial cell interactions may play previously unappreciated roles in the hormonal control of the primate reproductive tract. The lines of evidence that have drawn us to this conclusion will be reviewed.

Estrogen and progestin receptors in the reproductive tract of male and female primates.

The hamster uterine luminal epithelium undergoes cyclic morphological changes, which include mitosis and hypertrophy prior to ovulation and degenerative changes after ovulation. We have previously shown that the degenerative changes are symptoms of estradiol (E2) withdrawal; they occur within 24 hours after ovariectomy, can be prevented by E2 treatment, and are associated with a decline in the content of uterine nuclear E2 receptor (West et al., 1978).



In this report, the results of an electron microscopic study of the uterine luminal epithelium in cycling and hormonally altered hamsters are described. During estrus and after ovariectomy, the death of numerous epithelial cells occurs by apoptosis. The ultrastructural alterations in such cells include condensation and margination of the chromatin, cytoplasmic condensation, amoeboid changes in cell shape with formation of pseudopodial extensions, and nuclear and cytoplasmic fragmentation. The cell fragments, or apoptotic bodies, accumulate near the basal lamina, and are then phagocytosed by macrophages, and possibly by neutrophils as well.



The ultrastructural features of cell death by apoptosis in the hamster uterus are similar to those observed by others in a variety of tissues. Because of the periodic, hormonally regulated occurrence of extensive apoptosis in the hamster uterine epithelium during the estrous cycle, this system is well-suited for further investigations on the ultrastructural alterations that occur in cells undergoing apoptosis. In addition, apoptosis appears to be a sensitive morphological endpoint of E2 withdrawal in the hamster uterine luminal epithelium.

Hormonal control of apoptosis in hamster uterine luminal epithelium.

Monoclonal antibodies to the estrogen receptor (anti-ER) were used to develop an immunocytochemical method to detect ERs in frozen sections of the macaque reproductive tract. Specific nuclear, but not cytoplasmic, staining occurred with two different methods: direct, in which an antiestrophilin -horseradish peroxidase conjugate was used as the first antibody, and indirect, in which a mixture of antiestrophilins was used in the first incubation step. Nuclear staining was absent when various control antibodies replaced the anti-ER. In uteri from spayed monkeys treated with estradiol (E2) for 14 days, nuclear staining was always present. In uteri from similar animals treated for an additional 14 days with E2 and progesterone, nuclear staining was almost completely absent. Mean endometrial nuclear ER levels, measured by an exchange assay, were 5-fold greater in the E2-treated than in the E2-plus progesterone-treated group. In addition, when samples of estrogenized uterus and oviduct were incubated for 60 min in vitro with 100 nM E2, the intensity of nuclear staining increased in parallel with an increase in the concentration of nuclear ER. The nuclei of stromal, smooth muscle, and epithelial cells of the estrogenized oviduct, cervix, and vagina as well as smooth muscle cells of the estrogenized myometrium were also receptor positive. Nontarget tissues, such as duodenum, colon, esophagus, and skeletal muscle, contained no cells that showed specific nuclear staining. Some staining of cytoplasmic and extracellular components occurred in all preparations. These latter reactions were nonspecific, because they were present in many nontarget tissues or when control antibodies replaced the anti-ER. With current methods, only nuclear ERs can be reliably localized in frozen sections of monkey tissues with monoclonal antiestrophilins .

Immunocytochemical Localization of Estrogen Receptors in the Macaque Reproductive Tract with Monoclonal Antiestrophilins

Artificial menstrual cycles were producedin ovariectomized rhesus monkeys by a sequential estradiol-progesterone regimen. Cyclic changes in oviductal morphology and residual cytoplasmic estradiol binding capacity were correlatedwith systemic levels of estradiol and progesterone.The plasma estradiol levels averaged 200pg/ml throughout the entire experimental period.This level is characteristic of the late follicular phase in the rhesus monkey. Progesterone levels achieved during the artificial luteal phase (approximately 7 ng/ml) were similar to those produced by a functional corpus luteum. The cyclic changes in oviductal morphology were essentially identical to those found during natural cycles. Cilia developedand secretory activity began during the artificial follicular phase; deciliation occurred and secretory activity ceased during the artificial luteal phase.Thus, the effects of estradiol on the growth anddifferentiation of the oviduct were cyclically inhibitedby progesterone even though the estradiol...

Cyclic changes in oviductal morphology and residual cytoplasmic estradiol binding capacity induced by sequential estradiol--progesterone treatment of spayed Rhesus monkeys.

Ovarian androgens may act locally to modulate follicular and luteal function in various species. This study examined the distribution of androgen receptors within the primate ovary throughout the menstrual cycle. Ovaries were collected from rhesus and cynomolgus monkeys during the early, mid-, and late (n = 3-5 per stage) follicular and luteal phases of the cycle. The tissues were processed for indirect immunocytochemical localization of androgen receptors with a specific monoclonal antibody against human androgen receptor (AN1-15). In addition, ovaries (n = 3) were collected from rhesus monkeys for biochemical detection of androgen receptor using 3H-androgen and AN 1-15, Specific immunocytochemical staining, as determined by comparing adjacent tissue sections incubated with either AN 1-15 or a nonspecific control antibody, was exclusively nuclear. Androgen receptor was detected in the germinal epithelium and ovarian stroma at all stages of the cycle. The thecal and granulosa cells of growing follicles, and of many but not all atretic follicles, contained androgen receptors, Luteinizing granulosa cells of the periovulatory follicle and luteal cells from the early and midluteal phase stained intensely for androgen receptor. Regressing corpora lutes of the late luteal phase also stained for androgen receptor; however, fully regressed corpora lutes in the early follicular phase of the next cycle did not exhibit receptor staining. Luteal cells that were androgen receptor-positive also stained histochemically for the presence of 3�3-hydroxysteroid dehydrogenase. Sucrose gradient analysis with radiolabeled androgen demonstrated a shift in the androgen receptor peak in monkey ovarian tissue upon addition of ANI-15, confirming the presence of androgen receptor. These data support a receptor-mediated autocrine or paracrine role for androgen in the theca and granulosa layers during fofficular development in the primate ovary. Moreover, the evidence suggests for the first time a local androgen action within the corpus luteum during the menstrual cycle.

Neal B. West

Papers

Estrogen and progestin receptors in the reproductive tract of male and female primates.

Hormonal control of apoptosis in hamster uterine luminal epithelium.

Immunocytochemical Localization of Estrogen Receptors in the Macaque Reproductive Tract with Monoclonal Antiestrophilins

Cyclic changes in oviductal morphology and residual cytoplasmic estradiol binding capacity induced by sequential estradiol--progesterone treatment of spayed Rhesus monkeys.

Localization of androgen receptor in the follicle and corpus luteum of the primate ovary during the menstrual cycle.