Nicotinamide prevents the development of diabetes in the cyclophosphamide-induced NOD mouse model by reducing beta-cell apoptosis.

The development of diabetes in non-obese diabetic (NOD) mice, which normally takes between 3 and 7 months, can be accelerated by cyclophosphamide (CY) injections, with rapid progression to diabetes within only 2-3 weeks. This insulin-dependent diabetes mellitus (IDDM) can be prevented or delayed in CY-treated NOD mice by nicotinamide (NA). The present study was undertaken to determine the mode of cell death responsible for the development of IDDM in CY-treated male NOD mice and to investigate the effect of NA on beta-cell death. Apoptotic beta cells were present within the islets of Langerhans in haematoxylin and eosin-stained sections of the pancreata harvested from 3- and 12-week-old male NOD mice, from 8 h until 14 days after a single intraperitoneal injection of CY (150 mg/kg body weight). The maximum amount of beta-cell apoptosis in 3-week-old animals occurred 1-2 days after CY treatment (20 apoptotic cells per 100 islets), after which time levels of apoptosis declined steadily throughout the 14-day period studied. The incidence of beta-cell apoptosis in 12-week-old male NOD mice occurred in two peaks; the first was recorded 8-24 h after CY treatment (30 apoptotic cells/100 islets), while the second, at 7 days (36 apoptotic cells per 100 islets), coincided with increased insulitis. Administration of NA 15 min before CY treatment, and thereafter daily, substantially reduced the amount of apoptosis and effectively eliminated (4 apoptotic cells per 100 islets) the second wave of beta-cell apoptosis seen at day 7 in 12-week-old animals given CY alone. These results show that apoptosis is the mode of beta-cell death responsible for the development of CY-induced IDDM and that prevention of IDDM by NA is associated with a reduction in beta-cell apoptosis.

Expression and localization of the retinoblastoma gene during radiation-induced apoptosis in neonatal rat kidney.

An in vivo neonatal rat kidney model was used to study an association between expression and localization of the retinoblastoma tumor-suppressor gene (Rb), or its protein product (pRb), and localization of radiation-induced apoptosis. The rat kidney has two distinct zones of differentiation at birth-an outer nephrogenic zone, in which cells are undifferentiated and new nephrons are forming, and a differentiated zone internal to this zone that has essentially the adult kidney form. At 6 h after radiation (5 Gy), high levels of relatively synchronous apoptosis are induced in the nephrogenic zone, with little effect on the differentiated zone, and proliferation in the nephrogenic zone is almost totally inhibited by radiation treatment, again with little effect in the differentiated area. We have used our knowledge of this model to analyze control (sham-treated) and irradiated renal tissue for Rb mRNA transcript levels and localization (Northern blot and in situ hybridization (ISH)), pRb expression (Western blot and immunolocalization), apoptosis and mitosis (light and electron microscopy, and DNA gel electrophoresis for apoptosis), and cells in S-phase ([3H]thymidine uptake and autoradiography). Northern blots showed no detectable alteration in Rb transcript levels between control and irradiated tissues, whereas Western blots indicated increased expression of pRb in protein extracted from irradiated kidney compared with controls. ISH confirmed that Rb transcripts were not substantially altered in the nephrogenic and differentiated zones in control versus irradiated renal tissue. Immunolocalization of pRb demonstrated little effect in the differentiated zone, but in the nephrogenic zone pRb expression was increased, especially the S-shaped prenephrons, and was also found in many, but not all, apoptotic cells in this zone. The results link radiation-induced apoptosis and increased pRb expression in a zone of the neonatal kidney having a low level of cell differentiation.

Apoptosis is the mode of beta-cell death responsible for the development of IDDM in the nonobese diabetic (NOD) mouse.

The NOD/Lt mouse, a widely used model of human autoimmune IDDM, was used to establish the mode of beta-cell death responsible for the development of IDDM. Apoptotic cells were present within the islets of Langerhans in hematoxylin and eosin-stained sections of pancreases harvested from 3- to 18-week-old female NOD/Lt mice (a range of 11-50 apoptotic cells per 100 islets). Immunohistochemical localization of insulin to the dying cells confirmed the beta-cell origin of the apoptosis. Although some islets from age-matched control female NOD/scid mice contained apoptotic cells, virtually all of these cells were insulin negative as determined by immunohistochemistry. The small number of apoptotic insulin-positive cells identified in islets from NOD/scid mice (a range of 0-1 apoptotic cells per 100 islets) was not statistically significant, compared with the numbers recorded in NOD/Lt mice. All dying cells showed the morphological changes characteristic of cell death by apoptosis and stained positively with the TUNEL method for end-labeling DNA strand breaks. The maximum mean amount of beta-cell apoptosis occurring in NOD/Lt mice was at week 15 (50 apoptotic cells per 100 islets), which coincided with the earliest onset of diabetes as determined by blood glucose, urine glucose, and pancreatic immunoreactive insulin measurements. While there was no peak incidence of beta-cell apoptosis throughout the time period studied (weeks 3-18), the incidence of apoptosis decreased at week 18, by which time 50% of the animals had overt diabetes. The low levels of beta-cell apoptosis observed is indicative of a gradual deletion of the beta-cell population throughout the extensive preclinical period seen in this model and would be sufficient to account for the beta-cell loss resulting in IDDM. Apoptosis of beta-cells preceded the appearance of T-cells (CD3-positive by immunohistochemistry) in islets. Lymphocytic infiltration of islets (insulitis) was not detected until week 6. The results show that beta-cell apoptosis is responsible for the development of IDDM in the NOD/Lt mouse and that its onset precedes lymphocytic infiltration of the islets.

Apoptosis, mitosis, PCNA and bcl-2 in normal, leukoplakic and malignant epithelia of the human oral cavity: prospective, in vivo study.

Disordered balance between proliferation and apoptosis may contribute to carcinogenesis. Thirty-two oral biopsies were collected prospectively: 10 normal (N), 10 leukoplakia (dysplasia, D = 5; hyperplasia, H = 5) and 12 squamous cell carcinoma (C: 11). Distant normal tissue was also collected (HN, DN, CN). Based on counts of 1000 cells/slide, mitotic (MI), apoptotic (AI) and proliferating cell nuclear antigen (PCNA: PI) indices were calculated and bcl-2 expression recorded. AI correlated with MI (P < 0.001), but not PI or bcl-2 expression. PCNA was higher in H and HN than other groups (P < 0.0001). bcl-2 was reduced in C and CN (P < 0.001). Peak mitosis shifted basally in dysplasia, whilst peak apoptosis remained unaltered. These data confirm topographical alterations in proliferation relative to apoptosis in dysplasia of the oral cavity. Reduced bcl-2 in carcinoma and related 'normal' epithelium was unexpected, and may contribute to the high incidence of metachronous carcinomas in these patients.

Comparative alterations in p53 expression and apoptosis in the irradiated rat small and large intestine.

Temporal and spatial relationships between radiation-induced apoptosis and expression of p53 mRNA and protein were compared in rat small and large intestine. Apoptosis was quantified using morphological criteria, and p53 expression determined by immunohistochemistry or whole-tissue Northern analysis. In the small intestine, peak levels of apoptosis appeared earlier (4 h) than in the large intestine (6 h). p53 mRNA transcript levels in small and large intestine were not significantly altered from control levels at any time after treatment. However, in treated small and large intestine, cells showed increased positivity for p53 protein, increasing 10-fold over control levels 4-5 h after irradiation. A strong spatial relationship was found between high incidence apoptosis and p53 protein positivity. We compared published data of stem cell population positions for small and large intestine with our results. Target cells for apoptosis and p53 expression occurred at approximately fifth position from the crypt base of the small intestine, a zone coincident with stem cell population. Target cell position for apoptosis and p53 expression in the large intestine was again at fifth or sixth position from the base, but this zone is not the reported stem cell position (first or second position) for large intestine. Results from our model of radiation-induced intestinal apoptosis indicate that p53 protein is closely associated both temporally and spatially with the induction of apoptosis, and support the work of others in suggesting that p53 expression is modulated post-transcriptionally. Furthermore, our results support a hypothesis that apoptotic targeting of damaged stem cell populations, early response for apoptotic removal of DNA-damaged cells and/or early repair of these damage cells are all important parameters that determine differences in levels of tumorigenesis in the small and large intestine.

Expression and localization of clusterin mRNA in the small and large intestine of the irradiated rat: its relationship with apoptosis.

Apoptotic elimination of intestinal cells following irradiation has been studied in the small and large intestine of the rat, and correlated with the level of expression and localization of clusterin mRNA. Clusterin was moderately expressed in normal intestine where only small levels of apoptosis were found. After irradiation, however, there was a temporal correlation between an increased apoptotic index and increased clusterin expression. Localization of clusterin mRNA by in situ hybridization identified extensive labelling in the lower part (Paneth cell region) of small intestinal crypt, whereas epithelial cells in the large intestine were diffusely labelled. Clusterin expression was not localized over apoptotic cells and its role may be as a cell protection factor for surviving cells, as had been suggested by others. Clusterin may also be involved in remodelling of the intestinal crypt after radiation damage, a process that includes altering cell-to-cell contact, apoptosis, and sloughing of the dead cells from the intestinal villi. Our results do show a close temporal link between apoptosis and clusterin expression, and, as such, expression of the gene may be a useful indicator of presence of apoptosis in the irradiated intestine.

Beta-cell apoptosis is responsible for the development of IDDM in the multiple low-dose streptozotocin model.

Although insulin-dependent diabetes mellitus (IDDM) results from irreversible loss of beta cells, the mode of cell death responsible for this loss has not previously been categorized. In this study, the multiple low-dose streptozotocin (stz) model (intraperitoneal injection of stz at a concentration of 40 mg/kg body weight per day for five consecutive days) was used to investigate beta-cell death during the development of IDDM in male C57B1/6 mice. Apoptotic cells were evident by light microscopy within the islets of Langerhans of treated animals from day 2 (the day of the second stz injection) until day 17. Immunohistochemical localization of insulin to the dying cells confirmed the beta-cell origin of the apoptosis. Two peaks in the incidence of beta-cell apoptosis occurred: the first at day 5, which corresponded to an increase in blood glucose concentration, and the second at day 11, when lymphocytic infiltration of the islets (insulitis) was maximal. Insulitis did not begin until day 9, by which time treated animals had developed overt diabetes as revealed by blood glucose and pancreatic immunoreactive insulin (IRI) measurements. Beta-cell apoptosis preceded the appearance of T-cells in the islets and continued throughout the period of insulitis. Thus, whether induced by stz or a subsequent immune response, apoptosis is the mode of cell death responsible for beta-cell loss in the multiple low-dose stz model of IDDM.

Apoptosis in normal epithelium, premalignant and malignant lesions of the oropharynx and oral cavity: a preliminary study.

To explore the involvement of apoptosis in the development of oral and oropharyngeal squamous cell carcinoma (SCC) in vivo, biopsies were taken from patients with macroscopically normal (n = 6), leukoplakic (n = 12) or malignant (n = 8) mucosa. Leukoplakic lesions were divided histologically into dysplasia (n = 5) or carcinoma in situ (CIS: n = 7). Material was prepared for light and electron microscopy. The apoptotic index (AI), vertical cell position of apoptoses (cp), mitotic index (MI) and AI:MI ratio were calculated for each patient. AI increased from 0.12% +/- 0.07 S.E.M. (normal) to 0.58 +/- 0.13 (CIS) but fell to 0.14 +/- 0.14 in SCC. Apoptoses were suprabasal in normals, but generalised in CIS. MI increased from normal (0.20 +/- 0.06) to SCC (0.32 +/- 0.09), and AI:MI was at its maximum in CIS (1.57; SCC: 0.44). The results suggest that a change in apoptosis accompanies the onset of invasion in a premalignant lesion of the human oral cavity and oropharynx.

Calcium chelators induce apoptosis--evidence that raised intracellular ionised calcium is not essential for apoptosis.

A moderate sustained rise in intracellular ionised calcium has been observed to be associated with apoptosis occurring in many experimental systems. The application of extracellular and intracellular chelators of calcium has been reported to produce a decrease in apoptosis, while the addition of calcium ionophores often increases apoptosis. These findings, together with the observation of calcium-induced internucleosomal DNA cleavage in isolated nuclei, have suggested that DNA cleavage (and apoptosis) is a calcium-dependent process. However, a number of studies have shown that apoptosis is not always associated with a rise in the level of intracellular ionised calcium. In the present study, calcium chelators were found to induce apoptosis in cultured cells, concomitant with a decrease in both intracellular ionised calcium and total cell calcium content. Decreased intracellular ionised magnesium was also induced by extracellular chelators. These findings provide further evidence that a raised intracellular ionised calcium is not universally present during the induction of apoptosis. It is proposed that loss of calcium homeostasis, rather than a sustained rise in cytosolic calcium, is a determining factor in cell death by apoptosis.

Apoptosis in the human thymus in sudden and delayed death.

We counted apoptotic thymic cortical lymphocytes in semi-thin plastic sections of 266 human autopsy thymuses. The decreased patients included in the study were all aged less than 40 yrs and all died within 72 hrs of the onset of symptoms. Based on published data on animal and human material it was anticipated that individuals dying within 3 hrs of the onset of their fatal condition--"sudden deaths"--would have low apoptotic counts and those dying 3 to 72 hrs after the onset of their fatal condition--"delayed deaths"--would have elevated counts. Results showed that the sudden death group did have low apoptotic counts (mean 0.24 +/- 0.03% Standard Error of the Mean (SEM); n = 208) and the delayed death group did have significantly higher counts (mean 17.94 +/- 3.38% SEM; n = 58; p < 0.001). However, there were a number of cases in the latter group with low counts (n = 19). All of these patients had suffered injuries or clinical conditions that have been shown by others to be capable of interfering with the production of cortisol. As elevated cortisol is a major cause of apoptosis in thymic cortical lymphocytes, any such interference might prevent elevated apoptotic counts of thymic cortical lymphocytes.

Apoptosis. Its significance in cancer and cancer therapy.

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.

A flow cytometric study of cell death: failure of some models to correlate with morphological assessment.

The balance between cell death and cell proliferation is a significant factor in the growth kinetics of normal and neoplastic tissues. Distinction between the two major forms of cell death, necrosis and apoptosis, is now recognized as important in understanding mechanisms regulating cell survival. A recent approach in the study of apoptosis has been the use of flow cytometry, with some reports indicating that, when stained with propidium iodide (PI), the DNA of apoptotic cells has decreased fluorescence compared with that of viable cells. In this study, we investigated a flow cytometric procedure which used the simultaneous analysis of DNA content and 90 degrees light scatter (90LS). Significant differences in the PI staining pattern and a shift in 90LS were observed when apoptotic death occurred at different stages of the cell cycle. Importantly, such differences only allowed accurate quantification of apoptosis when it occurred in G1. While necrosis could be distinguished from apoptosis when examined during its early stages, a similar staining pattern to that found with apoptosis was observed when necrosis was examined during its latter stages. The results indicate that the measurement of DNA staining cannot be exclusively relied upon to detect apoptosis occurring in all models. However it is useful in the investigation of this process when the death occurs in G1, in that the method offers a rapid means for quantification.

Spontaneous apoptosis in NS-1 myeloma cultures: effects of cell density, conditioned medium and acid pH.

Apoptosis is a form of cell death which plays an important role in many biological processes including the regulation of B and T lymphocyte functions. We report here the spontaneous development of extensive apoptosis in cultures of the NS-1 mouse myeloma cell line following overgrowth. The apoptosis was identified by both its ultrastructural features and its DNA fragmentation pattern. High cell density and conditioned medium, but not acid pH, were found to be major inducers of apoptosis in this experimental system.

Apoptosis induced by microtubule disrupting drugs in cultured human lymphoma cells. Inhibitory effects of phorbol ester and zinc sulphate.

The effects of the microtubule disrupting drugs (MDD) vinblastine, vincristine and colchicine on a human lymphoma cell line, BM 13674, were investigated. Twelve hours after administration of vinblastine (10(-3) mg/ml), vincristine (10(-2) mg/ml) or colchicine (10(-2) mg/ml), cell death with the characteristic morphology of apoptosis was observed in 71.6%, 82.2% and 76.9% of the cells respectively. The mode of death was confirmed as apoptotic by the occurrence of internucleosomal DNA cleavage, which was demonstrated by agarose gel electrophoresis. For the purpose of casting light on the mechanism involved, inhibition tests were performed on apoptosis induced by one of these drugs, vinblastine, using a phorbol ester (PDBu), zinc sulphate and cycloheximide. PDBu, an activator of protein kinase C, and zinc sulphate, a putative inhibitor of the endonuclease were thought to be responsible for internucleosomal DNA cleavage; both markedly reduced the induction of apoptosis. The protein synthesis inhibitor cycloheximide, on the other hand, had no inhibitory effect. Moreover, cycloheximide treatment per se enhanced apoptosis. This suggests that new protein synthesis is not required for the execution of vinblastine-induced apoptosis. Such a finding is in accord with recent reports suggesting that the "death program" within many cell types may be primed but unable to proceed due to concomitant production of specific "apoptotic inhibitors". It is suggested that phorbol esters prevent vinblastine-induced apoptosis in the BM 13674 cells by activating one or more of these specific "apoptotic inhibitors", possibly by means of PKC-mediated phosphorylation.

Cell death induced by vincristine in the intestinal crypts of mice and in a human Burkitt's lymphoma cell line.

Although vincristine is widely used clinically in the treatment of some human cancers, its mechanism of action has not been clearly established. In this study, the patterns of cell death induced by vincristine in the intestinal crypts of mice and in a human Burkitt's lymphoma cell line were investigated by light and electron microscopy. Vincristine was found to enhance apoptosis of interphase cells in both systems and also to cause the arrest of cells in mitosis, the latter effect being more pronounced in the intestinal crypts. Arrested mitotic cells went on to die by a process that had a number of features in common with apoptosis. These include compaction of chromatin (following coalescence of chromosomes), condensation of the cytoplasm, initial preservation of organelle integrity, and eventually the fragmentation of the cell into a number of membrane-enclosed bodies which are morphologically similar to conventional apoptotic bodies. The results suggest that the cytocidal effect of vincristine is not solely dependent on metaphase arrest but is a cumulative one, resulting both from apoptosis of interphase cells and the 'apoptotic-like' death of cells arrested in metaphase.

Spermatogonial apoptosis has three morphologically recognizable phases and shows no circadian rhythm during normal spermatogenesis in the rat.

In this study we examined the possibility that regular or circadian fluctuations occur in the frequency of spontaneous spermatogonial apoptosis. Apoptosis of A2, A3 and A4 type spermatogonia occurring spontaneously in the normal rat testis was studied by light and electron microscopy. Normal and apoptotic A3 spermatogonia were quantified in 36 animals killed at two-hourly intervals over a 24 h period. Three sequential phases of spermatogonial apoptosis were defined and quantified separately: (i) an early phase in which cells showed margination of nuclear chromatin, (ii) an intermediate phase in which phagocytosed apoptotic bodies were partly degraded and (iii) a late phase in which only debris of degraded apoptotic bodies was evident. Groups of spermatogonia linked by intercellular bridges underwent apoptosis synchronously. Normal and apoptotic A3 spermatogonia occurred at a mean frequency of 33.4 and 9.6 per 10 seminiferous tubule profiles respectively; there was a large variation in these frequencies between animals, but no peaks or circadian periodicity were detected. Progressive degradation of apoptotic bodies was evident, the average ratios of intermediate and late bodies to early bodies being 1.5 and 3.5, respectively. Absence of a circadian rhythm in these data does not exclude the possibility that initiation of apoptosis in susceptible spermatogonial clones is synchronous, and that affected clones undergo lag periods of differing duration before expressing morphological apoptosis.