Protooncogenes as mediators of apoptosis.

Apoptosis has been well established as a vital biological phenomenon that is important in the maintenance of cellular homeostasis. Three major protooncogene families and their encoded proteins function as mediators of apoptosis in various cell types and are the subject of this chapter. Protooncogenic proteins such as c-Myc/Max, c-Fos/c-Jun, and Bcl-2/Bax utilize a synergetic effect to enhance their roles in the pro- or antiapoptotic action. These family members activate and repress the expression of their target genes, control cell cycle progression, and execute programmed cell death. Repression or overproduction of these protooncogenic proteins induces apoptosis, which may vary as a result of either cell type specificity or the nature of the apoptotic stimuli. The proapoptotic and antiapoptotic proteins exert their effects in the membrane of cellular organelles. Here they generate cell-type-specific signals that activate the caspase family of proteases and their regulators for the execution of apoptosis.

Induction of feline immunodeficiency syndrome by feline leukemia virus: pituitary and adrenocortical dysfunctions.

Seven young cats were injected with feline leukemia virus (FeLV); six of them became viremic. All of the viremic cats developed AIDS-related symptoms, i.e. lymphopenia, neutropenia, thymic atrophy, and wasting syndrome, along with an altered pituitary and adrenocortical function. These symptoms closely resemble human AIDS induced by HIV. It was discovered that, after 2 weeks of infection, the average amount of plasma adrenocorticotropic hormone (ACTH) detected in the infected cats was reduced by 29% in comparison with that before the infection. In contrast to the second week, the fifth week of infection showed a 94% increase of plasma ACTH which then dropped back down to 38% after the sixth and seventh weeks. This opposing biphasic pattern of change was also observed in the plasma cortisol content of the infected cats. The amount of change in plasma cortisol did not correlate with the detected increase in plasma ACTH, indicating a weak adrenal response to pituitary action.

Changes in thyroid-stimulating antibody activity in Graves' disease treated with antithyroid drug and its relationship to relapse: a prospective study.

Thyroid-stimulating antibodies (TSAb) were found in 51 patients (83.6%) with untreated Graves' disease. The mean TSAb index reached the normal level after 6 months of antithyroid drug therapy. In 9 patients (14.8%), TSAb remained persistently positive during and after antithyroid drug therapy, and relapse occurred in all. In 42 patients (68.8%), TSAb indices gradually returned to the normal range after drug treatment was started; in 38% of these, remission was maintained after treatment was stopped, in another 40.5%, relapse occurred when they were TSAb-positive, and the remaining 21.5% relapsed despite return of TSAb indices to the normal range. A positive TSAb index at the end of drug treatment was a useful indicator in predicting subsequent relapse because, with 1 exception, all patients who were still TSAb positive at drug withdrawal relapsed subsequently.

Down-regulation of insulin receptors in postnecrotic cirrhosis of liver.

Eighteen patients with postnecrotic cirrhosis of liver, including three patients who had had a portosystemic shunt operation, and 19 normal controls were studied. The tests performed included monocyte insulin receptor assay, iv glucose tolerance test, glucagon test, and insulin tolerance test. Insulin resistance was documented by the presence of fasting hyperglycemia and glucose intolerance together with hyperinsulinemia as well as resistance to exogenous insulin. The binding of [125I]insulin to monocyte insulin receptors was significantly decreased in cirrhotic patients compared with that in controls (P less than 0.02), and this was due to a significant decrease in the high affinity association constant (P less than 0.005). There was a significant negative correlation between the fasting insulin level and maximum [125I]insulin binding in cirrhotic patients (r = -0.8; P less than 0.02). Cirrhotics that had had a shunt operation showed a higher fasting insulin level, a greater insulin resistance, and a smaller maximum [125I]insulin binding to insulin receptors than those without shunt. All of these findings suggested a down-regulatory effect of hyperinsulinemia on the monocyte insulin receptors. An impaired glycemic response to glucagon was also found in cirrhotics, the exact mechanism of which remains to be elucidated. However, as the increases in plasma cAMP after glucagon were similar in cirrhotics and controls, the fault apparently did not lie in the glucagon receptor.

A prospective study of the changes in thyrotropin binding inhibitory immunoglobulins in Graves' disease treated by subtotal thyroidectomy or radioactive iodine.

The effects of subtotal thyroidectomy and radioactive iodine on thyroid-stimulating immunoglobulins, as measured by a receptor assay, more appropriately termed TSH binding inhibitory immunoglobulins (TBII), were studied in 74 patients with Graves' disease. Fourty-four patients received radioactive iodine therapy, while 30 were subjected to subtotal thyroidectomy. After radioactive iodine, more patients were TBII-positive (90.5% vs. 81.8%) than before treatment, and the mean TBII index decreased dramatically, the maximum decrease being at 3 months. The mean TBII index subsequently returned gradually to the pretreatment level. Subtotal thyroidectomy had a different effect on TBII activity. TBII indices were positive in 89.3% of these patients before any treatment but were positive in only 40% (12 patients) after antithyroid drugs had been given before surgery. After surgery, TBII indices remained positive in 7 patients, while the remaining 5 patients became TBII negative. Seventeen patients (56.7%) were TBII negative before operation and remained so after surgery. One patient who was TBII negative before operation became TBII positive 2 months after operation. Interestingly, postoperative relapse of hyperthyroidism occurred in 3 patients who were TBII positive, while hypothyroidism occurred in 7 patients who were TBII negative. Thus, the TBII activity after subtotal thyroidectomy might be an important factor in determining the outcome of surgery.

Autoimmunity in ocular and generalised myasthenia gravis.

Restricted ocular myasthenia gravis (OMG) and generalised myasthenia gravis (GMG) have been shown to differ in a number of respects. In OMG, antiacetylcholine receptor, antistriational and antinuclear antibodies were rare relative to their frequency in GMG. In contrast, antithyroid antibodies and a history of thyroid disease were much more prevalent in OMG than in GMG, OMG was not associated with the female predominance seen in GMG and appeared to be relatively common in some races rather than others. It is suggested that different pathogenetic mechanisms are responsible for these two forms of MG.

Thyrotropin receptors in adipose tissue, retro-orbital tissue and lymphocytes.

Thyrotropin (TSH) receptors on retro-orbital muscle and fat have been implicated in the pathogenesis of Graves' exophthalmos and it has been suggested that TSH has a direct effect on human fat metabolism. We have therefore investigated the interaction of biologically active 125I-labelled TSH with membranes prepared from human adipose, retro-orbital and thyroid tissue. Since lymphocytes contain receptors for several polypeptide hormones, TSH binding to lymphocyte membranes was also studied. We were unable to demonstrate TSH receptors in adult human adipose tissue, retro-orbital muscle and fat, or peripheral blood lymphocytes. In contrast, adult and neonatal guinea pig adipose tissue membranes showed similar TSH binding characteristics to guinea pig thyroid membranes.

Gossypol-induced apoptotic DNA fragmentation correlates with inhibited protein kinase C activity in spermatocytes.

After exposure of cultured rat spermatocytes to gossypol acetic acid for five hours, DNA fragmentation in a ladder pattern was found in the medium and supernatants of cell lysates. The concentrations of gossypol used for the induction of apoptosis ranged from 100 microM to 300 microM. Within this dose range, gossypol was also found to be effective at inhibiting protein kinase C (PKC) activity. This inhibitory effect was demonstrated by measuring the PKC residing in cytosolic and particulate fractions. However, the gossypol-induced inhibition of PKC activity was protected by phorbol 12,13-dibutyrate (PDBu), an activator of PKC. Furthermore, the presence of PDBu prevented gossypol-induced DNA fragmentation. These results suggest that spermatocyte apoptosis induced by gossypol is correlated with the reduction of PKC activity, and that maintenance of PKC basal activity is essential for protecting the spermatocyte from apoptosis.

Biological activity in the repopulating rat spermatocyte after the withdrawal of gossypol treatment. IV. The activity for the modification of core histones.

Mature male rats were treated with gossypol for 8 weeks. Afterwards, treatment was halted to allow the arrested spermatogonia to revive. Fifteen days after the withdrawal of the drug treatment, the repopulating pachytene spermatocyte (RPS) had a lower level of core histone H3 and H4 acetylation (40 and 55% reduction, respectively) than that of the control pachytene spermatocyte (CPS). The reduction in core histone acetylation was found in histone H3 and H4 but not in H2A and H2B. Forty-five days after the withdrawal of the drug treatment, the inhibitory effect on core histone acetylation was recovered. Both dot-blot and standard liquid assays were used to detect the nuclear histone acetylase activity in RPS and CPS. Fifteen days after the withdrawal of gossypol treatment, the acetylase type A activity in RPS was reduced by 42% when compared to CPS. It has been concluded that after gossypol treatment, the activity for nucleosomal core histones acetylation was selectively inhibited. This effect is related to an inhibitory effect on the histone acetylase activity.

PIP: Mature male rats were treated with gossypol for 8 weeks. Then treatment was halted to allow the arrested spermatogonia to revive. 15 days after the withdrawal of the drug treatment, the repopulating pachytene spermatocytes (RPS) had a lower level of core histone H3 and H4 acetylation (40 and 55% reduction, respectively) than that of the control pachytene spermatocytes (CPS). The reduction in core histone acetylation was found in histones H3 and H4, but not in H2A and H2B. 45 days after the withdrawal of the drug treatment, the inhibitory effect on core histone acetylation was recovered. Both dot-blot and standard liquid assays were used to detect the nuclear histone acetylase activity in RPS and CPS. 15 days after the withdrawal of gossypol treatment, the acetylase type A activity in RPS was reduced by 42% when compared to CPS. It was concluded that after gossypol treatment the activity for nucleosomal core histone acetylation was selectively inhibited. This effect is reacted to an inhibitory effect on the histone acetylase activity.

Biological activity in the repopulating rat spermatocyte after the withdrawal of gossypol treatment: III. The activity for the transcription of messenger RNA.

Mature male rats were treated with gossypol for 8 weeks after which the treatment was stopped to allow the arrested spermatogonia to revive. The arrested spermatogonia in the testes were allowed to develop for 15, 30, and 45 days. The amount of histone H2b, H4 and actin messenger RNA (mRNA) in the repopulated pachytene spermatocyte (RPS) and the control pachytene spermatocyte (CPS) was detected by the dot-blot hybridization technique. Histone H2b mRNA in total testicular RNA is higher than that detected in CPS and RPS. The amount of histone H2b mRNA in 5 micrograms to 10 micrograms of poly (A)+ RNA isolated from CPS is 235% higher than that detected in the RPS. The H2b mRNA content in the resting spermatocytes (15, 30, and 45 days after the withdrawal of gossypol treatment) was inhibited by 68, 72, and 45%, respectively. Histone H2b mRNA transcription in the resting spermatocyte of these 3 time periods was inhibited by 26, 40, and 0%, respectively. Fifteen days after the withdrawal of gossypol treatment, the amount of actin mRNA in RPS was inhibited by 47%. The inhibitory effect of gossypol was rapidly recovered within 30 days after the withdrawal of the drug treatment. It was concluded that after the withdrawal of gossypol treatment, the production of H2b, H4, and actin mRNA in RPS was inhibited. However, the inhibitory effect on the mRNAs' production is reversible. The recovery rate for the mRNAs follows the following order: Actin mRNA greater than H4 mRNA greater than H2b mRNA.

Biological activity in the repopulating rat spermatocyte after the withdrawal of gossypol treatment. V. Inhibition and recovery of microtubular dynein.

After oral administration with gossypol acetic acid for various times, young male rats developed a low content of microtubular (or cytoplasmic) dynein in the spermatogenic cells, e.g., spermatids and primary spermatocytes. The content of dynein in the cells was measured by enzyme-linked immunosorbent assay (ELISA) using monoclonal anti-dynein antibody. The results were expressed as ng dynein/10(6) cells and compared with those of the control rats. After gossypol treatment for 8, 12, and 19 weeks, the content of dynein in spermatids was reduced by 61%, 70%, and 68%, respectively; whereas, the amount of dynein in primary spermatocytes was reduced by 37%, 44%, and 31%, respectively. The microtubular dynein associated with spermatids was more vulnerable to gossypol than that of the primary spermatocytes. Immunofluorescent staining technique confirmed the finding that the control cells have more dynein than that of the drug-treated cells. Eight weeks after the withdrawal of the drug treatment, the content of dynein in spermatids and primary spermatocytes was fully recovered. The possible effects of this change in conjunction with the function of microtubules during spermatogenesis and sperm motility are discussed.

Reversible changes in the content of cellular and microtubular tubulin in spermatogenic cells after gossypol treatment.

After exposure of young male rats to gossypol acetic acid for various times, a reduction in the content of cellular and microtubular beta-tubulin was found in spermatocytes and spermatids. The content of tubulin was measured by enzyme-linked immunosorbent assay (ELISA). The results were expressed as micrograms tubulin/100 micrograms total protein and compared with those of the control rats. After drug treatment for 2, 6, 12, and 20 weeks, the content of total cell tubulin in spermatocyte was reduced by 2.4%, 8.8%, 52%, and 61%, respectively; whereas the content of tubulin in spermatid was reduced by 7.4%, 36%, 70%, and 72%, respectively. At the same time length of drug treatment, the content of microtubular tubulin in spermatocyte was reduced by 1.6%, 13%, 58%, and 61% in comparison to the reduction rate of 5%, 37%, 69%, and 77%, respectively, for spermatid. These results indicated that the tubulins associated with spermatids were more vulnerable to gossypol than that of the spermatocytes. Eight weeks after withdrawal of the drug treatment, the content of tubulin in spermatocytes and spermatids was recovered.

Reversible changes in the content of cellular and microtubular tubulin in spermatogenic cells after gossypol treatment.

After exposure of young male rats to gossypol acetic acid for various times, a reduction in the content of cellular and microtubular beta-tubulin was found in spermatocytes and spermatids. The content of tubulin was measured by enzyme-linked immunosorbent assay (ELISA). The results were expressed as micrograms tubulin/100 micrograms total protein and compared with those of the control rats. After drug treatment for 2, 6, 12, and 20 weeks, the content of total cell tubulin in spermatocyte was reduced by 2.4%. 8.8%. 52%, and 61%, respectively, whereas the content of tubulin in spermatid was reduced by 7.4%, 36%, 70%, and 72%, respectively. At the same time length of drug treatment, the content of microtubular tubulin in spermatocyte was reduced by 1.6% 13%, 58% and 61% in comparison to the reduction rate of 5%, 37%, 69%, and 77%, respectively, for spermatid. These results indicated that the tubulin associated with spermatids were more vulnerable to gossypol than that of the spermatocytes. Eight weeks after withdrawal of the drug treatment, the content of tubulin in spermatocytes and spermatids was recovered.

c-fos protein expression in apoptotic rat spermatocytes induced by gossypol.

Proto-oncogene products such as c-fos protein with a molecular weight of 62 kDa have been identified in rat spermatocytes. In this study, cellular levels of c-fos proteins in spermatocyte, either with or without gossypol exposure, were quantitatively detected by Western immunoblot and a computer-controlled Spot-denso-program with an IS-1000 Digital Imaging System. Within 0.5-3.5 h (an average of 2 h) of the addition of gossypol, levels of c-fos proteins fell dramatically. The reduction in c-fos proteins occurred 6 h before the apoptosis of spermatocytes in the presence of gossypol. Four hours after exposure to gossypol, the c-fos protein content was overexpressed. The period of c-fos up-regulation lasted for approximately 8 h. The increase in c-fos protein coincided with a high rate of apoptotic cell death. Morphologic structure of the dying cell was revealed by electron microscopy. These results suggest that spermatocyte apoptosis induced by gossypol correlates with biphasic c-fos protein-mediated apoptosis.

The effects of gossypol on nuclear protein in rat testes. I. Reduction in the content of basic proteins in the spermiogenic cells.

In rat testes, after 45 days of gossypol treatment, the number of round spermatids (RS) reduced from 9.8 x 10(6) to 6.2 x 10(6) per testis. The number of elongating spermatids (ES) reduced from 6.4 x 10(6) to 3.1 x 10(6) per testis. Total nuclear basic proteins (TNBP, i.e., somatic type of histones H1, H2A,B, H3, and H4, plus testis-specific proteins TP1,2,3, and sperm-specific S1 proteins) were extracted from RS and ES. Gossypol has no detectable effect on the concentration of TNBP in RS. However, significant effect was found in ES, where TNBP was reduced from 8.7 micrograms to 6.8 micrograms/10(6) cells. A two-stage polyacrylamide gel has been used to separate the TNBP and the individual proteins were quantified by a Beckman Model DU-7 computer-monitored spectrophotometer. In the RS cells, the content of the TNBP was not sensitive to gossypol. However, in the ES cells, the content of histones H1, H2A, and H2B were reduced by gossypol treatment (with an inhibition of 50%, 58%, and 31%, respectively). The inhibition on H3, TP1, TP3 and S1 was insignificant by gossypol.

The effects of gossypol on nuclear protein in rat testes. II. The synthesis of histones and testis-specific proteins after gossypol treatment.

This communication is to report the effects of gossypol on the synthesis of chromosomal basic proteins in the spermiogenic cells. A double-labelling experiment has been performed. The control rats received 14C-arginine, whereas the gossypol-treated rats received a 3H-arginine injection. An equal volume of the tissues (from control and gossypol-treated) were combined, and the total nuclear basic proteins (TNBP) from round spermatids (RS) and elongating spermatids (ES) were extracted for electrophoretic separation. By studying the ratio of 3H/14C, it was indicated that the synthesis of H1, H3, H2B plus H2A and TP1 in the RS was reduced by gossypol. The amount of inhibition of these proteins were 48%, 19%, 27%, and 11%, respectively. In the ES, the synthesis of H1, H3, H2B plus H2A, H4 plus TP2, TP3, and TP1 was reduced by gossypol, the reduction was 33%, 22%, 26%, 14%, 33%, and 8%, respectively. The synthesis of S1 protein was not inhibited by gossypol in both RS and ES.

Biphasic c-Myc protein expression during gossypol-induced apoptosis in rat spermatocytes.

The presence of protooncogene products such as c-Myc proteins in rat spermatocytes has been quantitatively detected by Western immunoblot and a computer-controlled Spotdenso-program with an IS-1000 digital imaging system. Cellular levels of c-Myc proteins in response to gossypol were measured in spermatocytes during the process of gossypol-induced apoptosis. Within 0.5 to 2 h of the addition of gossypol, levels of c-Myc proteins fall dramatically and remain at a low level for the next several hours. The reduction in c-Myc proteins occurs 4.5-6 h before the apoptosis of spermatocytes in the presence of gossypol. Between 3 and 5 h after exposure to gossypol, the c-Myc protein content returns to preexposure (or higher) levels. In addition, the increase in c-Myc proteins occurs 1.5-4 h before the apoptotic death of spermatocytes. An identical pattern of c-Myc protein response to gossypol was also found in total testicular tissue in vitro. These results suggest that spermatocyte apoptosis induced by gossypol is correlated with biphasic c-Myc protein expression. This article present some hypothetical models with which to explain c-Myc protein-mediated apoptosis.

Biological activity in the repopulating rat spermatocyte after the withdrawal of gossypol treatment. VI. Alteration in nuclear factors for interaction with histone gene promoter.

This article reports the effects of gossypol at the genomic level in rat spermatogenic cells. After gossypol treatment for various times (8, 12, and 19 weeks), the spermatogonial cells were allowed to rest for 2 to 4 weeks. The function of histone H4 gene promoter (H4GP) in the repopulating pachytene spermatocytes (RPS) was investigated. The sequences of the oligonucleotides for the H4GP binding sites 1 and 2 were synthesized by an ABI-392 DNA synthesizer. RPS and the control pachytene spermatocytes (CPS) were obtained by centrifugal elutriation and subsequently they were used for the preparation of nuclear protein extracts (NPE). The NPE interaction with the DNA fragment of site 1 or 2 was studied by an electrophoresis mobility shift assay (EMSA). EMSA with NPE-CPS revealed ten major gel shift bands for site 1 and 2. The presence of extra unlabelled DNA fragments competed with 6 of the bands. After 2 to 4 weeks recovery from 8, 12, and 19 weeks of gossypol treatment, NPE-RPS failed to shift four bands (b through e) in site 1. These results suggested that gossypol treatment affected the transcription factors for interaction with site 1. On the contrary, no effect was demonstrated in NPE that interacted with site 2. Furthermore, gossypol treatment did not change the nucleotide sequence in the H4GP site 1 and 2.

Biological activity in the repopulating rat spermatocyte after the withdrawal of gossypol treatment: II. The activity for the synthesis of cellular and nuclear basic proteins.

The synthesis of cellular and nuclear basic proteins in the first generation of the repopulated pachytene spermatocyte (RPS) and the control pachytene spermatocyte (CPS) was studied by the incorporation of 3H-arginine into proteins. After pretreatment with gossypol for various lengths of time, the synthetic activity for cellular protein in the RPS was slightly inhibited. In comparison to CPS, on the other hand, the inhibition ranged from 12% to 26% (for gossypol pretreatment from 3 to 12 weeks, respectively). However, the synthetic activity for nuclear basic protein in the RPS was drastically reduced in comparison to that of the CPS. The reduction ranged from 46% to 61% in RPS after receiving gossypol pretreatment for 3 to 12 weeks, respectively. Gel electrophoretic separation of the basic protein extracted from the pachytene cell indicated that the major basic proteins are nucleosomal linker and core proteins, i.e., histone H1, H2A,B, H3, and H4, and with a lesser amount of sperm-specific BP and X1 proteins. After gossypol treatment in the RPS, the synthesis of sperm-specific proteins (BP and X1) and core histones (H2A and H4) became drastically reduced. Finally, the effect of gossypol on the ratio of nuclear basic protein to DNA in RPS and the direct correlation of this ratio to nucleosomal spacing and chromatin structure are discussed.

Biological activity in the repopulating rat spermatocyte after the withdrawal of gossypol treatment: I. The activity for DNA synthesis.

Mature male rats were treated with gossypol for various lengths of time after which the treatment was stopped to allow the arrested spermatogonia to revive. Fifteen days after withdrawal of the drug treatment, the arrested spermatogonia entered into the meiotic division and then reached the pachytene stage. The meiotic cells derived from the arrested spermatogonia were the first generation of the differentiated germ cells after long-term gossypol treatment. Centrifugal elutriation technique was used to isolate the repopulating pachytene spermatocytes (RPS) or the control pachytene spermatocytes (CPS) from the rat testis with or without receiving gossypol pretreatment. In vitro culture condition for RPS and CPS was established for the synthesis of DNA by the measurement of 3H-thymidine incorporation. The activity for DNA synthesis in RPS was studied and compared to that of the CPS. It was concluded that after gossypol treatment for various times, the incorporation of 3H-thymidine into the cellular thymidine pool of RPS was not affected. However, the activity for DNA synthesis in RPS was significantly lower than that in the CPS. The synthetic activity for DNA was reduced by 14%, 26%, 42%, 40%, and 40% in the RPS for the gossypol pretreatment of 3, 4, 6, 7, and 8 weeks, respectively.