Biomedical subjects
L E Gerschenson
Publications and source records attributed to L E Gerschenson.
Early stages of p53-induced apoptosis are reversible.
Apoptosis is a type of physiological cell death that occurs during development, normal tissue homeostasis, or as a result of different cellular insults. The phenotype of an apoptotic cell is relatively consistent in most cases of apoptosis and involves at least changes in the cell membrane, proteolysis of cytoplasmic and nuclear proteins, and eventual destruction of nuclear DNA. Our laboratory is interested in the reversibility of apoptosis. We have initial evidence that DNA repair is activated early in p53-induced apoptosis and may be involved in its reversibility. The present work further strengthens our proposition that p53-induced apoptosis is reversible. We show that p53 activation induces phosphatidylserine (PS) externalization early in apoptosis, and that these early apoptotic cells with externalized PS can be rescued and proliferate if the apoptotic stimulus is removed. In addition, we show that unscheduled DNA synthesis occurs in early apoptotic cells, and that if DNA repair is inhibited by aphidicolin, apoptosis is accelerated. These results confirm that early p53-induced apoptotic cells can be rescued from the apoptotic program, and that DNA repair can modulate that cell death process.
The biology of apoptosis.
Apoptosis is a complex process that removes aging or injured cells from the body and occurs in a wide variety of organisms. Cell death has always been an integral aspect of the study of pathology, but only over the last 30 years or so has the interest in apoptosis gained appreciation in this field. This review analyzes pertinent aspects of apoptosis, from Virchow's initial descriptions of necrobiosis to more modern research, and reviews some of the key events and molecules involved in the process. Finally, the role of apoptosis in certain diseases and its importance in the clinical setting is addressed.
DNA repair is activated in early stages of p53-induced apoptosis.
p53 is a complex molecule involved in apoptosis, cell cycle arrest, and DNA repair. Since apoptosis may play an important role in deletion of neoplastic cells, an understanding of the mechanism of p53-induced apoptosis may be critical for possible future therapeutic interventions. Recent evidence suggests that p53-induced apoptosis may involve members of the nucleotide excision repair (NER) family, linking these two cellular events. Our work using a temperature-sensitive p53 construct further analyzes p53-induced apoptosis in cultured murine mammary epithelial cells and also suggests that DNA repair plays a role in that process. Although p21 is induced in our system, apoptosis occurs without a detectable preceding G1 cell cycle arrest and independent of cellular alterations brought on by the temperature shift. In addition, clonogenic assays suggest that early stages of p53-induced apoptosis may be reversible upon removal of the apoptosis stimulus. As a possible explanation for this reversibility, our results show that general DNA repair activity increases early in p53-induced apoptosis. We also show that caspase-3 is activated at a timepoint when colony formation begins to drop, suggesting a possible mechanism for the point of no return in p53-induced apoptosis.
Apoptosis: a different type of cell death.
Apoptosis is a type of cell death that plays an important role in early development and growth of normal adult tissues. It is regulated by physiological stimuli and is present in many species and tissues. The main morphological characteristics are nuclear fragmentation and cellular breakdown in apoptotic vesicles. Internucleosomal DNA fragmentation is an important biochemical feature that is the result of a yet to be isolated endonuclease activity. Experiments using RNA and protein synthesis inhibitors suggest the presence of either intracellular repressors or inducers of apoptosis.
Characterization of uterine epithelium apoptotic cell death kinetics and regulation by progesterone and RU 486.
The authors show here that progesterone suppresses apoptosis, and its antagonist RU 486 induces it in rabbit uterine epithelium, as assessed by morphologic and biochemical studies. The authors' studies demonstrate that internucleosomal DNA fragments are identifiable as early as 24 hours after ovariectomy of pseudopregnant rabbits, and become undetectable 6 days after ovariectomy. Maximal levels of DNA fragmentation (about 74% of total isolated DNA) were observed 36 hours after ovariectomy. The number of apoptotic cells appeared to increase parallel to the increased DNA breakdown, and accounted for approximately 26% of the uterine epithelial cells at 48 hours after ovariectomy. Levels of progesterone in serum dropped precipitously 6 hours after ovariectomy and remained very low for several days. Administration of progesterone, more than any other steroid hormone, to pseudopregnant ovariectomized rabbits, prevented the increase in apoptotic cell death. By contrast, administration of the anti-progestin RU 486 to pseudopregnant rabbits triggered apoptosis, which attained levels similar to those observed in ovariectomized animals. The authors' findings establish that uterine epithelium apoptosis occurs in a time-dependent fashion and provides strong evidence that the actions of progesterone in that tissue are not only to stimulate cell proliferation and differentiation, but also to suppress apoptosis.
Coordinated regulation of apoptosis and cell proliferation by transforming growth factor beta 1 in cultured uterine epithelial cells.
Cell and tissue growth is regulated through a complex interplay of stimulatory and inhibitory signals. We describe two biological actions of transforming growth factor beta 1 (TGF-beta 1) in primary cultures of rabbit uterine epithelial cells: (i) inhibition of cell proliferation and (ii) a concomitant increase in cells undergoing apoptosis (programmed cell death). It is proposed that proliferation and apoptosis together comprise normal cell growth regulation.
Prostaglandin photoaffinity probes: synthesis and biological activity of azide-substituted 16-phenoxy- and 17-phenyl-PGF2 alpha prostaglandins.
The development of a prostaglandin PGF2 alpha photoaffinity probe led to the synthesis and biological evaluation of azide-substituted 17-phenyl-18,19,20-trinorprostaglandin F2 alpha and 16-phenoxy-17,18,19,20-tetranorprostaglandin F2 alpha derivatives. Two approaches for the preparation of iodinated versions of these prostaglandins were evaluated: (1) iodination of a phenyl azide bearing an activating hydroxyl group and (2) iodination of an aniline precursor to the phenyl azide group and subsequent conversion of the aniline to the phenyl azide. In the first approach, 17-(4-azido-2-hydroxyphenyl)-18,19,20-trinorprostaglandin F2 alpha, 16-(5-azido-3-hydroxyphenoxy)-17,18,19,20-tetranorprostaglandin F2 alpha, and 16-(4-azido-2-hydroxyphenoxy)-17,18,19,20-tetranorprostaglandin F2 alpha were prepared by using the Corey synthesis, but were biologically inactive presumably as a result of the hydrophilic phenolic hydroxyl group. In the second approach, the iodination of a 17-(4-aminophenyl)-18,19,20-trinorprostaglandin F2 alpha derivative delivered 17-(4-azido-3-iodophenyl)-18,19,20-trinorprostaglandin F2 alpha, which exhibited competitive binding with natural [3H]PGF2 alpha to ovine luteal cells and to plasma membranes of bovine corpora lutea. [125I]-17-(4-Azido-3-iodophenyl)-18,19,20-trinorprostaglandin F2 alpha was utilized in a preliminary photoaffinity cross-linking experiment.
Biochemical evidence for programmed cell death in rabbit uterine epithelium.
Uterine epithelial cell proliferation, differentiation, and death are known to be regulated by estrogen and progesterone. The authors investigated a specific pattern of cell death called apoptosis, or programmed cell death, which is biochemically characterized by a specific pattern of DNA degradation. DNA isolated from endometrium of ovariectomized pseudopregnant rabbits showed a pattern of DNA cleavage at internucleosomal locations. In comparison, DNA from the endometrium of non-ovariectomized animals, as well as several other organs, did not exhibit that pattern. This biochemical evidence supports previous and present morphologic data and correlates with it. Under the experimental conditions used, only the uterine epithelial compartment of the endometrium shows apoptotic cell death, which is absent in the stromal compartment.
A role for prostaglandins in estrogen growth regulation.
Several lines of evidence support the theory that estrogen increases prostaglandin F2 alpha (PGF2 alpha) receptor number and amplifies the intracellular response to the uterine mitogen, PGF2 alpha, in primary cultures of rabbit endometrium. It is proposed here that estrogen induction of growth in target tissues may be mediated through prostaglandins. Determination of prostaglandin receptor status and prostaglandin levels in estrogen dependent tumors could be of importance as diagnostic tools. The use of prostaglandin synthesis inhibitors and/or antagonists in the therapy of these tumors should be considered.
Effect of phorbol ester on prostaglandin regulation of proliferation in rabbit endometrial cells.
We have proposed that two of the endogenously synthesized endometrial prostaglandins, prostaglandin F2 alpha (PGF2 alpha) and prostaglandin E1 (PGE1), play a regulatory role in growth control of the endometrium. PGF2 alpha increases DNA synthesis and PGE1 inhibits that effect. Primary cultures of rabbit endometrial cells were used here to examine the effects of the tumor-promoting, diacylglycerol mimicking, phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (TPA), on the prostaglandin control of cell proliferation. TPA treatment of these cultures results in: a decrease in control levels of proliferation and complete inhibition by TPA of PGF2 alpha stimulated DNA synthesis; a reduction in [3H]PGF2 alpha binding with short term treatment but an increase to above control binding level with long term treatment; an inhibition of the normal PGF2 alpha stimulated inositol polyphosphate synthesis; and a small increase in accumulation of PGF2 alpha in the culture media. Furthermore, in this culture system, TPA does not down regulate [3H]PGE1 binding; it does not alter the normal PGE1 stimulation of cAMP synthesis; and it has no effect on the normal endogenous PGE1 synthesis by these cultures. The above results are consistent with our previous observations that PGF2 alpha works through the intracellular messengers inositol polyphosphate/diacylglycerol whereas PGE1 works through cAMP.
Requirement for prostaglandin F2 alpha in 17 beta-estradiol stimulation of DNA synthesis in rabbit endometrial cultures.
We have hypothesized that two of the endogenously synthesized endometrial prostaglandins (PGs), prostaglandin F2 alpha (PGF2 alpha), and prostaglandin E1 (PGE1), play a regulatory role in growth control of the rabbit endometrium. PGF2 alpha increases DNA synthesis and PGE1 inhibits that effect. Primary cultures of rabbit endometrial cells were used to examine the possible role of these PGs in the mechanism of action of 17 beta-estradiol on DNA synthesis. Towards this end, binding, second messenger and DNA synthesis experiments were performed. 17 beta-estradiol stimulation resulted in a time dependent (optimal: approximately 6 h) and 17 beta-estradiol concentration dependent (optimal: approximately 10(-7) M 17 beta-estradiol in phenol red-containing medium) increase in [3H]PGF2 alpha binding. Scatchard type analysis of the binding data revealed an increase in receptor number while the receptor affinity for [3H]PGF2 alpha remained the same as in the control treated cultures. This 17 beta-estradiol stimulated increase in PGF2 alpha receptor allowed a suboptimal concentration of PGF2 alpha (10(-9) M) to increase intracellular levels of inositol polyphosphates, while by itself this concentration of PGF2 alpha caused no significant change in intracellular inositol polyphosphate levels. 17 beta-estradiol, alone among the several studied steroid hormones, could increase [3H]PGF2 alpha binding. Proliferation studies revealed that, in these primary cultures of rabbit endometrium, 17 beta-estradiol could increase DNA synthesis but not in the presence of indomethacin, unless PGF2 alpha was added to the medium at a concentration (10(-10) M) near or above what is normally accumulated in the medium by these cultures. In the absence of 17 beta-estradiol stimulation, addition of these same low concentrations of PGF2 alpha had no effect on DNA synthesis. Apparently, through its effect on the PGF2 alpha receptor, 17 beta-estradiol enhances the PGF2 alpha stimulated DNA synthesis response approximately 100 fold. The DNA synthesis induced by 17 beta-estradiol can be inhibited by PGE1, as can PGF2 alpha-induced DNA synthesis. We propose that 17 beta-estradiol may be mediating its mitogenic effect through an alteration of the prostaglandin agonist:antagonist control of proliferation in rabbit endometrial cultures. In addition we suggest that, if 17 beta-estradiol acts to increase PGF2 alpha, receptors as part of its mode of action, this may be of importance in other tissues possessing both prostaglandin and 17 beta-estradiol receptors.
Uteroglobin production in the pseudopregnant rabbit uterus. Immunohistochemical studies.
Uteroglobin, a secretory protein of rabbit uterine epithelium, was localized by the direct immunoperoxidase method in control and hCG-induced pseudopregnant rabbits. In control rabbits, uteroglobin was confined to the apical cytoplasm of nearly all cells of the endometrial epithelium. The induction of pseudopregnancy resulted in a pronounced continuing increase, through 4 days post-hCG administration, in the absolute number of epithelial cells engaged in uteroglobin synthesis. Furthermore, the endoplasmic reticulum was more intensely stained for uteroglobin than in the epithelial cells of control rabbit endometrium. Thus, the increased production of uteroglobin, in response to hormonal stimulation, appears to be achieved both through an increase in the amount of uteroglobin synthesized by a given cell as well as by an increase in the number of cells involved in uteroglobin synthesis. Concurrent with the increase in the number of cells synthesizing uteroglobin, an increase in the number of unstained cells first appeared at the second day of pseudopregnancy, during the period of maximal epithelial proliferation. However, within those cells containing uteroglobin on the second day following injection with hCG, most staining was limited to the perinuclear membrane. At various times following hCG administration, a number of scattered cells, intensely stained for uteroglobin, were observed in the uterine epithelium. Based upon ultrastructural studies, failure to exclude trypan blue, and the presence of intra-mitochondrial uteroglobin, they were identified as either dead or dying cells.
Estradiol-17 beta and progesterone regulate secretion of uteroglobin through different pathways.
Uteroglobin, the primary secretory protein of rabbit uterine epithelium, was localized by the direct immunoperoxidase method in uteri of control ovariectomized rabbits and of ovariectomized rabbits injected with progesterone or estradiol-17 beta. In control rabbits, staining for uteroglobin was almost entirely abolished six weeks after bilateral ovariectomy. Two days following progesterone injection of ovariectomized rabbits, intense staining for uteroglobin could be detected within the endoplasmic reticulum, Golgi complexes, and compact secretory vesicles of most endometrial epithelial cells. Estradiol-17 beta injection resulted in a different intracellular pattern of uteroglobin distribution. Two days following treatment with that steroid hormone, intense staining for uteroglobin was localized within large apical mucous droplets and moderate staining was present in the endoplasmic reticulum and Golgi complexes of these cells. The increased mucin content of the endometrial epithelium following treatment with estradiol-17 beta was confirmed by a periodic acid-Schiff histochemical reaction in the presence of diastase. Quantitation by radioimmunoassay of uteroglobin production in vitro by uterine fragment confirmed that progesterone had a greater effect on enhancing uteroglobin production than estradiol-17 beta and that both steroid hormones did not have any effect after 30 min of incubation in vitro. We suggest that progesterone not only regulates uteroglobin production at the transcriptional level, but that it also regulates the mode of uteroglobin secretion by the induction of a different pathway, compared with the one used when estradiol-17 beta is administered alone.
Hormonal regulation of cell death in rabbit uterine epithelium.
It is known that estrogen (E) and progesterone (P) play important roles in the regulation of endometrial growth. In the rabbit endometrial epithelium, a balance is maintained between cell proliferation and cell death which seems to be under ovarian hormonal control. In this study the authors determined cell proliferation by quantitating the mitotic index (MI) and cell death by quantitating the death index (DI) in uterine histologic sections from whole animals that were hormone treated versus control rabbits. E caused proliferation of uterine epithelial cells and decreased the DI transiently, while P also increased proliferation but decreased the DI dramatically. In a time course study, after a single injection of human chorionic gonadotropin to induce pseudopregnancy, there was transient decrease in the DI and an increase in the MI between Days 2 and 5. In pseudopregnant animals, hormones had no effect in intact animals, but after ovariectomy there was about a 124-fold increase in the DI, which could be prevented by P administration. The predominant type of cell death observed in this system is apoptosis (97.5%), as opposed to necrosis (2.5%). Thus, it is proposed that cell death may be as important as cell proliferation in the regulation of normal uterine epithelial growth.
Prostaglandin F2 alpha and E1 regulation of proliferation in primary cultures of rabbit endometrial cells.
The study of growth of endometrial cells is of importance in reproductive biology. Several factors and hormones are thought to play important roles in the control of growth. Prostaglandin F2 alpha (PGF2 alpha) causes an increase in both tritiated thymidine ([3H]Tdr) incorporation into DNA and in the cell number of primary cultures of rabbit endometrial cells cultured in a serum-free, chemically defined media. Prostaglandins F1 alpha, E1, E2, A2, and B2 and arachidonic acid (all tested at 10(-7) M) do not affect [3H]Tdr incorporation as compared to control cultures. The increase in [3H]Tdr incorporation into DNA in response to PGF2 alpha stimulation is concentration-dependent (optimal approximately 3 X 10(-7) M) and is seen starting approximately 9 hr poststimulation. Both prostaglandin E1 (PGE1) and prostaglandin E2 (PGE2), but not PGs F1 alpha, I2, A2, B2, their parent molecules, or related molecules, antagonize and can completely block the PGF2 alpha-induced increase in [3H]Tdr incorporation into DNA. This antagonism is seen both when the cells are pretreated with PGE1 prior to the PGF2 alpha stimulation and when the cells are exposed to both PGE1 and PGF2 alpha simultaneously. Exogenously added 8-Br-cAMP mimics the PGE1 antagonism of PGF2 alpha. The PGF2 alpha-induced increase in [3H]Tdr incorporation is not synergistic, antagonistic, or additive with the [3H]Tdr incorporation increase in response to either estradiol-17 beta or epidermal growth factor. The specific effect of PGF2 alpha on primary culture endometrial cell growth and its antagonism by PGE1, PGE2, and 8-Br-cAMP are new findings.
Binding and second messengers of prostaglandins F2 alpha and E1 in primary cultures of rabbit endometrial cells.
Several factors and hormones are thought to play a role in the growth control of endometrial cells. We have shown that prostaglandin F2 alpha (PGF2 alpha) is a growth factor for primary cultures of rabbit endometrial cells grown in serum-free, chemically defined medium and that prostaglandin E1 (PGE1) antagonizes the PGF2 alpha induction of growth (Orlicky et al., 1986). [3H]PGF2 alpha binds to whole cells in a time (optimal approximately 30 min)- and temperature-dependent (optimal 37 degrees C), disassociable (90% disassociable within 30 min), saturable (Kd1 = 4.9 X 10(-8) M, n1 = 1.2 X 10(5) molecules/cell; Kd2 = 2.6 X 10(-7) M, n2 = 3.0 X 10(5) molecules/cell), and specific manner. [3H]PGE1 binds in a time-dependent (optimal 25 min), disassociable (90% disassociable within 10 min), saturable (Kd = 6.4 X 10(-8) M, n = 1.2 X 10(5) molecules/cell), and specific manner. This specific binding of [3H]PGF2 alpha and [3H]PGE1 is down-regulatable by prior treatment of the cultures with unlabeled ligand, and up-regulatable by prior treatment of the cultures with indomethacin to inhibit endogenous PG synthesis. Proteolytic enzyme treatment for 2 min reduces the specific binding of PGF2 alpha by 75%. PGE1 stimulates intracellular cAMP synthesis and accumulation in a time (optimal 10 min)- and concentration (half-maximal stimulation at 10(-6) M)-dependent manner but has no effect on intracellular cGMP. PGF2 alpha has no effect on either intracellular cAMP or cGMP in this system. We describe here for the first time the analysis at a biochemical level of the interaction between two prostaglandins, antagonistic to each other in terms of growth regulation.
Regulation of inositol phosphate levels by prostaglandins in cultured endometrial cells.
Stimulation of cultured rabbit endometrial cells by one of the rabbit endometrial cell culture proliferation factors, prostaglandin F2 alpha (PGF2 alpha), resulted in a very rapid increase in the intracellular levels of [3H]-inositol triphosphate (IP3), [3H]-inositol biphosphate (IP2), and [3H]-inositol monophosphate (IP1) in cells prelabeled with [3H]-inositol. These increases in inositol phosphate levels were detected in periods of stimulation as short as 30 seconds, reached a maximum by 1 1/2-2 min and declined to control levels by 6-10 min. The stimulation was dose-dependent with maximal increases observed near 10(-6) M PGF2 alpha. The cholinergic agent, carbachol, also led to time and dose-independent increases in IP3. Lithium, cadmium, silver, copper, and zinc ions had no effect either on the breakdown of IP3 or on the accumulation of IP1. In contrast, vanadate at 10(-6) or 10(-5) M did lead to a decrease in the breakdown of IP1 and a concomitant increase in IP1, IP2, and IP3. PGF2 alpha was found previously to induce an increase in rabbit endometrial cell DNA synthesis which was inhibited by concomitant or prior addition of prostaglandin E1 (PGE1). PGE1, in a dose-dependent manner, was found to inhibit the observed IP3 increase by PGF2 alpha at 1 1/2 min of stimulation. PGF2 alpha treated and control cultures did not differ in cAMP or cGMP levels, cellular 45Ca uptake, nor cellular 22Na uptake. We propose that IP3 may be one of the intracellular messenger(s) synthesized following the treatment of rabbit endometrial cell cultures with the proliferation agent PGF2 alpha and that it may play a crucial role with cAMP in growth regulation.