DNA Nick-end Labeling Research Articles

Apoptosis and expression of apoptosis-related genes in the mouse testis following heat exposure

Objective: To investigate molecular mechanisms of germ cell apoptosis induced by heat exposure in mice. Design: Controlled laboratory study. Setting: Departments of Urology and Biochemistry, Yamagata University School of Medicine, Yamagata, Japan. Animal(s): Forty-four male B6D2F1 mice. Intervention(s): Heat exposure, 43°C for 15 minutes. Main Outcome Measure(s): Testicular germ cell apoptosis (percentages of apoptotic tubules and apoptotic cells) was assessed by using DNA nick-end labeling, and expression of Bcl-2 family, Fas-FasL system, and p53 was evaluated by using Western analysis. Result(s): Bilateral testicular weights decreased significantly from 3 days after heat exposure. Percentages of apoptotic tubules and apoptotic germ cells increased significantly from 1 day after heat exposure. There were no significant changes in the levels of Bcl-xl, Bad, and Bax after heat exposure. However, Bcl-2 expression level decreased significantly 7 days after heat exposure. In contrast, the expression level of Fas and p53 increased significantly from 1 day to 3 days after heat exposure, respectively. Expression level of FasL elevated significantly at days 1 and 2 but declined from day 3. Conlusion(s): Germ cell apoptosis induced by heat exposure is mainly mediated by the Fas-FasL system.

Early massive follicle loss and apoptosis in heterotopically grafted newborn mouse ovaries.

Ovarian tissue cryopreservation and transplantation can be used to restore fertility to sterile females. A question that warrants further investigation is whether the follicular content is affected by the freeze-thawing and grafting procedure, and if so, to what extent and by what mechanism. Intact newborn mouse ovaries were allografted under the kidney capsule or were cryopreserved by slow freezing with dimethylsulphoxide as the cryoprotectant prior to grafting. Estrogenic activity of ovariectomized recipient mice, as revealed by vaginal cytology, resumed after 11 days of transplantation. At 14 days after transplantation, ovarian grafts were recovered and processed histologically for follicle number counting. The follicular content of grafts of fresh ovaries was 58% of that from ovaries of age-matched 14 day old mice. In frozen-thawed ovarian grafts, the follicular content was only 9% lower than that of fresh grafted ovaries. Apoptosis of follicular cells was investigated by DNA nick end labelling. We observed a marked increase in the staining of fragmentation of DNA shortly after transplantation (2-12 h) of fresh newborn mouse ovaries. The results of the present study indicate that transplantation rather than cryopreservation accounts for the major and early loss of primordial follicles in grafted newborn mouse ovaries.

Apoptosis index and apoptosis-related antigen expression in serrated adenoma of the colorectum: the saw-toothed structure may be related to inhibition of apoptosis.

Serrated adenoma of the colorectum is a recently proposed entity characterized by a saw-toothed structure of hyperplastic polyp and cytologic atypia of tubular adenoma. To clarify the role of apoptosis in morphogenesis of serrated adenoma, we investigated apoptotic indices and expression of apoptosis-related antigens in the tumor cells. Thirty-eight serrated adenomas were examined by the nick-end DNA labeling method and immunostained for CD95 (Fas), bcl-2, bax, and p53. Thirty-seven hyperplastic polyps, 48 tubular adenomas, and 16 sections containing normal colonic mucosa were similarly examined for comparison. The apoptotic indices in the upper and middle zones of the crypts of serrated adenomas and hyperplastic polyps were lower than those of normal colon mucosa and tubular adenomas with statistically significant differences. The CD95 expression was diffusely observed throughout the epithelium of normal crypts and tubular adenomas, whereas it was reduced in serrated adenomas and hyperplastic polyps. The bcl-2 expression was confined to the basal crypts in the latter two lesions but was diffuse throughout the neoplastic epithelium in tubular adenomas. The bax expression was increased in serrated adenomas and tubular adenomas but was decreased in hyperplastic polyps. Overexpression of p53 protein was observed in 50% of serrated adenomas, none of hyperplastic polyps, and 14% of tubular adenomas. These findings suggest that inhibition of apoptosis is caused by reduced CD95 expression in serrated adenomas and hyperplastic polyps, which may induce the characteristic saw-toothed structure in these lesions. Based on the similarities and differences between serrated adenoma and hyperplastic polyp observed in the present study, a progression from the latter to the former lesion may be postulated.

Cytoplasmic c-Jun N-terminal immunoreactivity: a hallmark of retinal apoptosis.

1. We investigated the association of c-Jun with apoptosis within retinal tissue. Explants of the retina of neonatal rats were subject to a variety of procedures that cause apoptosis of specific classes of retinal cells at distinct stages of differentiation. The expression of c-Jun was detected by Western Blot, and immunohistochemistry was done with antibodies made for either N-terminal or C-terminal domains of c-Jun, and correlated with apoptosis detected either by chromatin condensation or by in situ nick end labeling of fragmented DNA. 2. c-Jun protein content was increased in retinal tissue subject to induction of both photoreceptor and ganglion cell death. 3. c-Jun N-terminal immunoreactivity was found mainly in the cytoplasm of apoptotic cells regardless of cell type, of the stage of differentiation, including proliferating cells, or of the means of induction of apoptosis. 4. The data are consistent with the hypothesis that c-Jun is involved in the control of cell death in retinal tissue, but other proteins that cross-react with c-Jun N-terminal antibodies may also be major markers of retinal apoptosis. 5. Antibodies directed to c-Jun N-terminal (aa 91-105) are useful tools to follow apoptotic changes in retinal tissue.

Overexpression of Copper/Zinc Superoxide Dismutase in Transgenic Rats Protects Vulnerable Neurons against Ischemic Damage by Blocking the Mitochondrial Pathway of Caspase Activation

Mitochondria are known to be involved in the early stage of apoptosis by releasing cytochrome c, caspase-9, and the second mitochondria-derived activator of caspases (Smac). We have reported that overexpression of copper/zinc superoxide dismutase (SOD1) reduced superoxide production and ameliorated neuronal injury in the hippocampal CA1 subregion after global ischemia. However, the role of oxygen free radicals produced after ischemia/reperfusion in the mitochondrial signaling pathway has not been clarified. Five minutes of global ischemia was induced in male SOD1-transgenic (Tg) and wild-type (Wt) littermate rats. Cytosolic expression of cytochrome c and Smac and activation of caspases were evaluated by immunohistochemistry, Western blot, and caspase activity assay. Apoptotic cell death was characterized by DNA nick end and single-stranded DNA labeling. In the Wt animals, early superoxide production, mitochondrial release of cytochrome c, Smac, and cleaved caspase-9 were observed after ischemia. Active caspase-3 was subsequently increased, and 85% of the hippocampal CA1 neurons showed apoptotic DNA damage 3 d after ischemia. Tg animals showed less superoxide production and cytochrome c and Smac release. Subsequent active caspase-3 expression was not evident, and only 45% of the neurons showed apoptotic DNA damage. A caspase-3 inhibitor (N-benzyloxycarbonyl-val-ala-asp-fluoromethyl ketone) reduced cell death only in Wt animals. These results suggest that overexpression of SOD1 reduced oxidative stress, thereby attenuating the mitochondrial release of cytochrome c and Smac, resulting in less caspase activation and apoptotic cell death. Oxygen free radicals may play a pivotal role in the mitochondrial signaling pathway of apoptotic cell death in hippocampal CA1 neurons after global ischemia.

In vitro evidence that cortisol directly modulates stress-related responses in the skin epidermis of the rainbow trout (Oncorhynchus mykiss Walbaum)

Exposure of fish to stressors leads to multiple changes in the skin epithelium. We investigated the role of the stress hormone cortisol in the control of these changes by exposure of pieces of skin from the rainbow trout Oncorhynchus mykiss with an in vitro tissue culture incubation procedure. The effects of 24 h exposure to 4 cortisol concentrations (0, 50, 500 and 1000 ng/ml) were determined. Numbers of mucous, mitotic and apoptotic cells were quantitatively assessed using immunohistochemical techniques, in situ DNA nick end labelling (TUNEL), as well as conventional light and scanning electron microscopy (SEM). The cortisol receptor blocker mifepristone was used to investigate whether the effects could be attributed to the direct action of the hormone via glucocorticoid receptors. Overall, cortisol had no effect on the mucous cell population at 24 h. Incubation with the receptor blocker reduced the number of mucous cells. Cell proliferation was stimulated by the addition of 50 and 500 ng/ml cortisol, but not at 1000 ng/ml. Incubation with the receptor blocker increased proliferation in the control group (0 ng/ml) only. An increase in apoptosis occurred at 500 and 1000 ng/ml cortisol. This increase was blocked by incubation with the receptor blocker, which resulted in lower numbers of apoptotic cells in all except the 0 ng/ml controls. SEM observations corroborated the quantitative data. The results indicated that the effects of stressors on the fish epidermis mentioned above are mediated by cortisol, with the exception of mucous cell release.

Human Telomerase Reverse Transcriptase, p53 and Ki-67 Expression and Apoptosis in Colorectal Serrated Adenoma

Background: Serrated adenoma (SA) has recently been proposed as a distinct histological lesion of the colorectum. However, no definite histopathologic criteria for SA have been established, and its histogenesis and natural history remain unclear. Methods: We analysed 25 hyperplastic polyps (HPs), 26 low-grade SAs (LG-SAs), 32 high-grade SAs (HG-SAs), 18 low-grade tubular adenomas (LG-TAs), 16 high-grade TAs (HG-TAs) and 20 carcinoma in situ (CIS). To clarify molecular features of SA, we used in situ hybridization to examine the expression of human telomerase reverse transcriptase (hTERT), immunohistochemistry to examine the expressions of p53 and Ki-67, and in situ DNA nick end labeling to detect apoptotic cells. Results: The incidence of hTERT expression was 1 (4.0%) of 25 for HP, 12 (46.2%) of 26 for LG-SA, 18 (56.3%) of 32 for HG-SA, 6 (33.3%) of 18 for LG-TA, 7 (43.8%) of 16 for HG-TA, 12 (80.0%) of 15 for CIS, respectively. The incidence of hTERT expression in SA was significantly higher than that in HP. Seventeen (29%) of the 58 SAs were regarded as positive for p53 protein, but none of the HPs showed p53 immunoreactivity. Ki-67 labeling index in SA, TA and CIS was significantly higher than that in HP. The apoptototic index was not significantly different between HP, SA, TA and CIS. In HG-SA, the incidence of hTERT expression in p53-positive lesions was significantly higher than that in p53- negative lesions. Conclusions: These results suggest that hTERT and p53 expression increase in the early stages of carcinogenesis in SA and that SA has a malignant transformation similar to that of TA. It may be useful to investigate hTERT and p53 expression for differential diagnosis of SA from HP.

Induction of lymphocyte apoptosis in rat liver allograft with ongoing rejection by FTY720.

The action mechanism of FTY720, a novel immunosuppressant, is completely different from conventional immunosuppressants. The drug, which triggers apoptosis in murine and human lymphocytes, has a potent immunosuppressive activity to prevent allograft rejection without any severe side-effect. The present study was designed to determine whether FTY720 induces apoptotic cell death in activated lymphocytes infiltrated into liver grafts with ongoing rejection. FTY720 was orally administered at 5 mg/kg to the recipients on day 3 and day 4 after grafting, when the graft rejection was histologically confirmed. The intragraft patterns of IL-2, interferon-gamma (IFN-gamma), perforin, and granzyme B gene expression were detected by reverse transcriptase-polymerase chain reaction. The treatment reversed ongoing rejection and significantly prolonged recipient survival time compared with the control group. Light microscopic observation of the graft sections stained with the DNA nick-end labelling method showed that the apoptosis in the control allografts was mainly induced in hepatocytes, while that in the FTY720-treated allografts was in infiltrated lymphocytes. The rejection therapy with FTY720 did not alter the expression of IL-2, IFN-gamma, and perforin mRNAs, but slightly decreased granzyme B expression. Our results suggest that FTY720 does not alter the intrinsic lymphocyte function to produce the rejection-related cytokines, but strongly induces apoptotic cell death in the activated lymphocytes. Thus, FTY720 affords new insight into the mechanisms underlying improvements in immunosuppressive treatments.

Nitric oxide and cyclic guanosine monophosphate stimulate apoptosis via activation of the Fas-FasL pathway.

Inappropriately exaggerated response of pulmonary vascular cells to inflammatory mediators may be one mechanism that leads to acute (or adult) respiratory distress syndrome. Nitric oxide (NO) is induced following such exaggerated responses and may have a variety of biological effects, including induction of apoptosis. The mechanism by which NO causes apoptosis is unknown; however, Fas (CD95) and Fas ligand (FasL) (CD95L) have been implicated. We hypothesized that NO-induced apoptosis in pulmonary vascular smooth muscle cells is mediated through a Fas-FasL pathway. Cultured human and rat pulmonary artery smooth muscle cells (PASMCs) were exposed to soluble FasL (0-5 ng/ml), the NO donor S(G)-nitroso-N-acetyl pencillamine (SNAP) (0-50 microg/ml), and/or anti-FasL (0-100 microg/ml) for 12 h. Apoptosis was measured using in situ DNA nick end labeling and flow cytometry. Changes in Fas and FasL protein levels were assessed via Western blot analysis. Messenger RNA (mRNA) abundance of apoptosis-related genes was determined using a ribonuclease protection assay. Rat PASMCs exposed to FasL show a dose-dependent increase in apoptosis. Human PASMCs are less responsive to FasL. Addition of anti-FasL to rat PASMCs treated with 10(-5) M SNAP decreases apoptosis levels compared to SNAP treated alone. FasL and Fas receptor proteins are increased in response to 10(-3) to 10(-4) M SNAP or 10(-6) M 8-bromo-cyclic guanosine monophosphate (cGMP). The mRNA abundance of Fas, FasL, and other apoptosis-related genes is increased in response to 10(-6) M 8-bromo-cGMP but not 8-bromo-cyclic adenosine monophosphate. Nitric oxide-induced apoptosis in rat and human PASMCs is mediated, at least in part, through the Fas-FasL pathway, with cGMP increasing the expression of Fas and FasL.

Enhanced apoptosis and radiosensitization by combined 13-CIS-retinoic acid and interferon-α2a; role of RAR-β gene

Purpose: Combined use of 13-cis-retinoic acid (cRA) and interferon-α2a (IFNα) induced significant radiosensitization in human cervical cancer ME-180 cell line, whereas it failed to achieve similar radiation enhancement in HeLa cells. The differential radiosensitization could be from the difference of retinoic acid receptor (RAR) expression because RAR-β was highly expressed in ME-180 cells in contrast to the HeLa cells where RAR-β was not detectable. We examined the role of this gene in mediating radiosensitization by cRA and IFNα, and explored the mechanism of radiation-induced cell killing.Methods and Materials: Human cervical cancer cell lines, ME-180 and HeLa, were treated with cRA and IFNα followed by radiation. Apoptosis and radiosensitization were quantitated by TUNEL assay (in situ DNA nick end labeling) and colony-forming ability of surviving cells. The cells were transfected with bcl-2 gene and RAR-β gene to test the role of these genes in mediating radiosensitization and apoptosis.Results: Synergistic radiosensitization and apoptosis was observed by combined use of cRA and IFNα with radiation in ME-180 cells which express high level of RAR-β mRNA, whereas these were not seen in HeLa cells where RAR-β mRNA is not detectable. Both radiosensitization and apoptosis were abolished by bcl-2 gene in ME-180 cells. RAR-β gene transfection induced similar radiation enhancement and apoptosis in HeLa cells.Conclusion: Apoptosis and radiation response were enhanced in the cells with high level of RAR-β mRNA expression. The RAR-β gene appears to mediate the radiation-induced apoptosis by cRA and IFNα. These findings indicate that presence of RAR-β in the cancer cells could be exploited for patient selection in using these drugs for apoptosis and radiosensitization.

Expression of fas/fas ligand (fasL) and its involvement in infiltrating lymphocytes in hepatocellular carcinoma (HCC).

This study was conducted to examine the expression of Fas/Fas ligand (FasL), to elucidate its relationship with tumor-infiltrating lymphocytes (TILs), and to detect possible gene mutation of Fas/FasL in patients with hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). Indirect immunohistochemical staining was performed on formalin-fixed, paraffin-embedded sections of liver biopsy and surgery specimens from five normal livers, and from the livers of 30 patients with HCC. Fas/FasL mRNA-expressing cells and apoptotic cells were detected by in situ hybridization and DNA nick end labeling (TUNEL), respectively. We also performed polymerase chain reaction (PCR)-amplifying and direct sequencing for the Fas/FasL gene. Fas/FasL and its mRNA were localized on the membrane or in the cytoplasm in some HCC cells, as well as hepatocytes. Their expression was enhanced in areas with infiltrating inflammatory cells in the noncancerous regions of liver tissue and on the margins of the cancerous tissue. The positivity rate for TUNEL was elevated along these margins. The labeling index of Fas/FasL was lower in the cancerous liver tissue than in the surrounding noncancerous region (P < 0.01), and tended to decrease in proportion to the malignancy of tumor cells; Fas/FasL expression was not found on poorly differentiated type cancer cells. Fas(-)/FasL(+), FasL-mRNA(+) HCC cells were seen in one specimen of moderately differentiated type. Some CD8+T lymphocytes were TUNEL-positive around the cancerous region. In this study, cancerous and noncancerous tissues in HCC revealed no genetic mutations in any exons of Fas/FasL. These findings suggest that Fas/FasL expression was decreased in proportion to the malignancy of tumor cells, and that infiltrating CD8+ T lymphocytes play a role in apoptosis in HCC. The apoptosis in HCC could be regulated by the suppression of Fas/FasL expression, or, sometimes, by the enhancement of FasL expression.

Ebselen reduces cytochrome c release from mitochondria and subsequent DNA fragmentation after transient focal cerebral ischemia in mice.

The seleno-organic compound ebselen has both antioxidant and anti-inflammatory properties. Although ebselen has been shown to protect the brain against stroke, it is unclear how ebselen provides neuroprotection. In the present study the authors examined whether ebselen inhibits neuronal apoptosis resulting from transient focal cerebral ischemia in mice. The cytochrome c release and DNA fragmentation, both of which are biochemical markers of apoptosis, were compared between vehicle- and ebselen-treated mice. Cerebral ischemia was induced by transient middle cerebral artery occlusion for 30 minutes in ICR mice under halothane anesthesia. Ebselen (10 mg/kg) was given orally twice, 30 minutes before ischemia and 12 hours after reperfusion. By Western blot analysis, we examined release of mitochondrial cytochrome c. To evaluate brain damage, the brain sections were treated for terminal deoxynucleotidyl transferase-mediated DNA nick-end labeling (TUNEL) and Nissl staining. Prolonged neuroprotective efficacy of ebselen was determined by counting neuronal nuclei (NeuN) immunopositive cells at 21 days after ischemia. - Cytochrome c release was detected in the ischemic hemisphere at 3 to 24 hours after ischemia. Ebselen treatment diminished the cytochrome c release at 12 and 24 hours. In addition, ebselen decreased both DNA fragmentation determined by TUNEL and brain damage volume at 3 days after ischemia. Furthermore, ebselen increased the number of NeuN immunopositive cells at 21 days after ischemia. These results indicate that ebselen attenuates ischemic neuronal apoptosis by inhibiting cytochrome c release. Ebselen may be a potential compound in stroke therapy.

Albumin overload induces apoptosis in LLC-PK1cells

The degree of albuminuria is a well-known adverse prognostic indicator in human glomerular diseases. However, the mechanisms by which albuminuria by itself contributes to tubulointerstitial injury and progression of renal disease remain unclear. We tested the hypothesis that apoptosis may represent one of the mechanisms by which tubule epithelial cells are damaged after albumin overload in vitro. Cultured LLC-PK(1) proximal tubule cells were incubated with varying concentrations of BSA. This resulted in a dose- and duration-dependent induction of apoptosis, as evidenced by internucleosomal DNA cleavage (DNA laddering and nick-end labeling), externalization of plasma membrane phosphatidylserine (annexin labeling), and characteristic morphological changes (cell shrinkage and nuclear condensation). Albumin overload also resulted in a dose-dependent upregulation of Fas and Fas-associated protein with death domain (FADD), and activation of caspase 8. Incubation with the caspase 8 inhibitor IETD ameliorated the albumin-induced apoptosis. Collectively, our results indicate that albumin overload induces apoptosis of cultured LLC-PK(1) cells, mediated at least in part by the Fas-FADD-caspase 8 pathway.

Role of the increase in p21 in cisplatin-induced acute renal failure in rats.

The goal of this study was to clarify the role of p21, a cyclin-dependent kinase inhibitor, in acute renal failure (ARF). This was accomplished with the examination of the renal expression of p21 in cisplatin (CDDP)-induced ARF and in rechallenge injury with CDDP. The injection of CDDP (5 mg/kg) into rats induced increases in serum creatinine and tubular damage and the number of in situ DNA nick end labeling-positive cells, which peaked at day 5, followed by recovery to control levels by day 14. The rechallenge with the same dose of CDDP 14 d after the first dose of CDDP induced significantly less injury and no significant increase in in situ DNA nick end labeling-positive cells. The first CDDP dose significantly increased p53-positive nuclei at day 1, which disappeared by day 5, and the number of p21-positive nuclei, which had two peaks on days 3 and 9. The number of proliferating cell nuclear antigen (PCNA)-positive nuclei peaked at days 3 and 12. A significant increase in the incorporation of 5-bromo 2'-deoxyuridine (BrdU) was found at day 5 and peaked at day 7. The second injection of CDDP induced significant increases in the number of p21-, p53-, and PCNA-positive nuclei within 2 d but did not affect the incorporation of BRDU: These findings suggested that (1) CDDP induced two peaks of the increase in p21; (2) the first peak occurred shortly after CDDP and was accompanied by overexpression of p53 and PCNA but not with BrdU incorporation, possibly reflecting G1 arrest and DNA repair; (3) the second peak of p21 occurred through an p53-independent pathway and may contribute to cell differentiation; and (4) the overexpression of p21 and PCNA in rechallenge injury may contribute to acquired resistance in CDDP-induced ARF via enhanced DNA repair.

Glucocorticoid hormone can suppress apoptosis of rat testicular germ cells induced by testicular ischemia

Objective: To evaluate the effect of dexamethasone, a potent synthetic glucocorticoid hormone, on apoptosis of testicular germ cells and vascular neutrophil adhesion after repair of testicular torsion in rats. Design: Controlled laboratory study. Setting: Department of Urology, Yamagata University School of Medicine, Yamagata, Japan. Animal(s): Fifty-one male Sprague-Dawley rats. Intervention(s): Dexamethasone, 10 mg/kg of body weight. Main Outcome Measure(s): Testicular germ-cell apoptosis (percentages of apoptotic tubules and apoptotic cells) and vascular neutrophil adhesion were assessed by using DNA nick-end labeling and the endothelial-neutrophil adhesion score, respectively. Result(s): Intravenous administration of dexamethasone at repair of 90-minute testicular torsion significantly inhibited testicular germ-cell apoptosis and vascular neutrophil adhesion. This inhibition was suppressed by intravenous injection of mifepristone, a glucocorticoid-receptor antagonist. Conclusion(s): Glucocorticoids can be administered for torsion in addition to conventional torsion repair.

Expression of Fas ligand and apoptosis of disc cells in herniated lumbar disc tissue.

An examination of surgically obtained herniated lumbar disc tissues performed by using immunohistochemical staining and the DNA nick end labeling method. To investigate the cell type that expresses Fas ligand (FasL) and any evidence of apoptosis of the disc cells in herniated lumbar disc tissues. The Fas/FasL system is involved in delivering a death signal that rapidly commits the cells to apoptosis. In the authors' previous study, the expression of Fas on disc cells was identified in herniated lumbar disc tissue. Twenty-three herniated lumbar disc tissues (contained disc, n = 9; noncontained disc, n = 14) were examined to investigate the cell type that expresses FasL and any evidence of apoptosis of the disc cells by using immunohistochemical staining and the DNA nick end labeling method, respectively. The percentage of FasL-positive disc cells was calculated and compared with clinical and radiologic data. FasL was expressed in the cytoplasm of the disc cells, and nuclear DNA fragmentation in a few disc cells was identified. A higher degree of FasL expression in disc cells was found in noncontained discs than in contained discs (P < 0.05). The percentage of FasL-positive disc cells significantly increased with the patient's age (P < 0.05), but not with the degree of disc degeneration (P > 0.05). The current results indicate that disc cells, after herniation, undergo apoptotic cell death via autocrine or paracrine FasL mechanisms by the disc cells themselves.

Expression of MAGE antigens and analysis of the inflammatory T-cell infiltrate in human seminoma.

The MAGE gene family encodes antigens that are recognized by cytotoxic T-cells. The expression of MAGE antigens has been linked to tumor stage, and MAGE peptides are under investigation as possible vaccines. Seminomas are tumors that are typically accompanied by a heavy inflammatory infiltrate, but have not been studied with regard to their MAGE antigen expression and its correlation with the inflammatory infiltrate. We investigated, therefore, MAGE protein expression, the amount of cytotoxic T-cells, clonality of the lymphocytic infiltrate, apoptotic activity and occurrence of necrosis. Specimens of 27 patients with classical seminoma were examined by immunohistochemistry for CD4, CD8, CD56, CD45R0, beta2-microglobulin and HLA-DR. MAGE expression was detected with the monoclonal antibody 57B, reactive with MAGE-1, -3, -4, -6 and -12. Clonality of the inflammatory infiltrate was examined by multiplex polymerase chain reaction (PCR) analysis of the T-cell receptor rearrangement. Apoptotic cells were detected by DNA nick-end labeling of fragmented DNA, and the apoptotic index was determined semi-quantitatively. Expression of 57B was found in 19 (70%) of 27 seminomas. In all cases, more than 70% of T-cells expressed CD45R0. In four cases, a predominant infiltration of CD8-positive cytotoxic T-cells (CD4/CD8 ratio < 1) was present. However, 15 seminomas showed a CD4/CD8 ratio > 1. In all cases, infiltration of CD56-positive natural killer cells was only focal. HLA-DR expression was not detectable in tumor tissue; beta2-microglobulin was only focal in three cases. Analysis of the T-cell clonality revealed a polyclonal population. The apoptotic index was not significantly different in 57B-positive seminomas (4.15%) compared with 57B negative seminomas (3.80%). Also, no correlation between the 57B expression and the occurrence of necrosis was found. MAGE antigens are homogeneously expressed in most seminomas, but their presence does not appear to represent a dominant epitope responsible for the lymphocytic infiltrate.

Apoptosis and proliferation as predictors of chemotherapy response in patients with breast carcinoma

BACKGROUND Laboratory evidence suggests that many anticancer agents exert their effect by altering the ratio between apoptosis and cellular proliferation. The objective of this clinical study was to examine in vivo changes in apoptotic index (AI), Bcl-2 expression, proliferation (Ki-67 and S-phase fraction [SPF]), and ploidy as potential indicators of chemoresponsiveness. METHODS Twenty-eight women with primary operable breast carcinoma received 2M (mitoxantrone 11 mg/m2, methotrexate 35 mg/m2 every 3 weeks) for 4 cycles before surgery/radiotherapy, and an additional 2 cycles were given after surgery. Clinical response was assessed after four cycles of treatment according to World Health Organization criteria. Changes in molecular markers were assessed from biopsies obtained by fine-needle aspiration performed before treatment, repeated after 24 and/or 72 hours (T1), and on Days 7 and 21 after the first cycle of chemotherapy. Flow cytometric analysis was used to assess SPF, ploidy, and AI (in situ DNA nick end labeling assay) whereas Ki-67 and Bcl-2 were evaluated by immunocytochemical analysis. RESULTS The overall response rate was 61% (17 of 28 patients), with a 14% (4 of 28 patients) complete response rate. Patients with diploid carcinomas (P = 0.04) with high Ki-67 (P = 0.0001) and SPF (P = 0.09) were more likely to respond to treatment. Median AI increased by 3.4% with interquartile (IQ) range of 3.2 in responders, compared with only −0.1% (IQ range, 2.2) in nonresponders at T1 (P = 0.03). Median Ki-67 decreased by −12.0% (IQ range, 22.9) in responders and increased by 18.5% (IQ range, 15.1) in nonresponders on Day 21 (P = 0.003). Median Bcl-2 scores increased by 1.0 (IQ range, 4.0) in responders and were unchanged at 0.0 (IQ range, 0.5) in nonresponders (P = 0.08). Changes in SPF or ploidy were not significantly predictive of response. CONCLUSIONS The results of this preliminary study support evidence that chemotherapy may increase apoptosis, decrease Ki-67, and increase Bcl-2 expression in primary breast carcinomas that subsequently respond to therapy. Methodology allowing morphological confirmation of apoptosis would be advantageous. Cancer 2000;89:2145–52. © 2000 American Cancer Society.

Larval-to-adult conversion of a myogenic system in the frog, Xenopus laevis, by larval-type myoblast-specific control of cell division, cell differentiation, and programmed cell death by triiodo-L-thyronine

Abstract For the clarification of larval-to-adult muscle conversion, the authors established primary culture methods for adult- and larval-type myoblasts in the frog, Xenopus laevis, and examined the hormonal response in each case. The cell types were enzymatically dissociated from adult frog leg and tadpole tail muscles, respectively. The cells became attached to culture plates, proliferated, and fused with each other to form multinucleated myotubes within one week. Five significant differences between the two cell types were noted. (1) Adult cells showed greater proliferation activity than larval cells, the former increasing 5.5-fold over 6 days while the latter increase only 2.5-fold. (2) Differentiation (fusion) of larval type myoblasts started earlier. Cell fusion began on day 2 or 3 in larval cells and on day 4 in adult cells. (3) The metamorphic hormone, triiodo-L-thyronine (T3) decreased larval cell numbers to 56 % of that of control-cultures on day 7 but had no effect on adult cell number. DNA synthetic activity (3H-thymidine incorporation) in larval cells decreased under T3 (10− 8M) to 45 % of the control level on day 7. (4) Differentiation of adult myoblasts into myotubes was promoted by T3, whereas that of larval cells diminished by half. (5) Myotube death was induced by T3 specifically in larval but not in adult cultures. In addition to the myotube death, double staining with TUNEL (in situ DNA nick end labeling) and anti-desmin antibody indicated that T3 induces myoblast (desmin+ cell) death specifically in larval but not in adult cells. It is thus evident that the conversion of a larval-type myogenic system during metamorphosis becomes possible through nearly totally specific control of cell division, cell differentiation, and programmed cell death at a precursor cell level by T3.

Larval-to-adult conversion of a myogenic system in the frog, Xenopus laevis, by larval-type myoblast-specific control of cell division, cell differentiation, and programmed cell death by triiodo-L-thyronine

For the clarification of larval-to-adult muscle conversion, the authors established primary culture methods for adult- and larval-type myoblasts in the frog, Xenopus laevis, and examined the hormonal response in each case. The cell types were enzymatically dissociated from adult frog leg and tadpole tail muscles, respectively. The cells became attached to culture plates, proliferated, and fused with each other to form multinucleated myotubes within one week. Five significant differences between the two cell types were noted. (1) Adult cells showed greater proliferation activity than larval cells, the former increasing 5.5-fold over 6 days while the latter increase only 2.5-fold. (2) Differentiation (fusion) of larval type myoblasts started earlier. Cell fusion began on day 2 or 3 in larval cells and on day 4 in adult cells. (3) The metamorphic hormone, triiodo-L-thyronine (T 3) decreased larval cell numbers to 56 % of that of control-cultures on day 7 but had no effect on adult cell number. DNA synthetic activity ( 3H-thymidine incorporation) in larval cells decreased under T 3 (10 − 8 M) to 45 % of the control level on day 7. (4) Differentiation of adult myoblasts into myotubes was promoted by T 3, whereas that of larval cells diminished by half. (5) Myotube death was induced by T 3 specifically in larval but not in adult cultures. In addition to the myotube death, double staining with TUNEL (in situ DNA nick end labeling) and anti-desmin antibody indicated that T 3 induces myoblast (desmin + cell) death specifically in larval but not in adult cells. It is thus evident that the conversion of a larval-type myogenic system during metamorphosis becomes possible through nearly totally specific control of cell division, cell differentiation, and programmed cell death at a precursor cell level by T 3.