Accurate population-based model for individual prediction of colon cancer recurrence.

BACKGROUND: Prediction models are useful tools in the clinical management of colon cancer patients, particularly when estimating the recurrence rate and, thus, the need for adjuvant treatment. However, the most used models (MSKCC, ACCENT) are based on several decades-old patient series from clinical trials, likely overestimating the current risk of recurrence, especially in low-risk groups, as outcomes have improved over time. The aim was to develop and validate an updated model for the prediction of recurrence within 5 years after surgery using routinely collected clinicopathologic variables. MATERIAL AND METHODS: A population-based cohort from the Swedish Colorectal Cancer Registry of 16,134 stage I-III colon cancer cases was used. A multivariable model was constructed using Cox proportional hazards regression. Three-quarters of the cases were used for model development and one quarter for internal validation. External validation was performed using 12,769 stage II-III patients from the Norwegian Colorectal Cancer Registry. The model was compared to previous nomograms. RESULTS: The nomogram consisted of eight variables: sex, sidedness, pT-substages, number of positive and found lymph nodes, emergency surgery, lymphovascular and perineural invasion. The area under the curve (AUC) was 0.78 in the model, 0.76 in internal validation, and 0.70 in external validation. The model calibrated well, especially in low-risk patients, and performed better than existing nomograms in the Swedish registry data. The new nomogram's AUC was equal to that of the MSKCC but the calibration was better. CONCLUSION: The nomogram based on recently operated patients from a population registry predicts recurrence risk more accurately than previous nomograms. It performs best in the low-risk groups where the risk-benefit ratio of adjuvant treatment is debatable and the need for an accurate prediction model is the largest.

Targeted resequencing of candidate genes using selector probes.

Targeted genome enrichment is a powerful tool for making use of the massive throughput of novel DNA-sequencing instruments. We herein present a simple and scalable protocol for multiplex amplification of target regions based on the Selector technique. The updated version exhibits improved coverage and compatibility with next-generation-sequencing (NGS) library-construction procedures for shotgun sequencing with NGS platforms. To demonstrate the performance of the technique, all 501 exons from 28 genes frequently involved in cancer were enriched for and sequenced in specimens derived from cell lines and tumor biopsies. DNA from both fresh frozen and formalin-fixed paraffin-embedded biopsies were analyzed and 94% specificity and 98% coverage of the targeted region was achieved. Reproducibility between replicates was high (R(2) = 0, 98) and readily enabled detection of copy-number variations. The procedure can be carried out in <24 h and does not require any dedicated instrumentation.

Sustained TGF beta exposure suppresses Smad and non-Smad signalling in mammary epithelial cells, leading to EMT and inhibition of growth arrest and apoptosis.

To better understand the dual, tumour-suppressive and tumour-promoting function of transforming growth factor-beta (TGFbeta), we analysed mammary epithelial NMuMG cells in response to short and long-term TGFbeta exposure. NMuMG cells became proliferation-arrested and apoptotic after exposure to TGFbeta for 2-5 days, whereas surviving cells underwent epithelial-mesenchymal transition (EMT). After chronic TGFbeta exposure (2-3 weeks), however, NMuMG cells became resistant to proliferation arrest and apoptosis, showing sustained EMT instead (TD cells). EMT was fully reversed by a pharmacologic TGFbeta-receptor-I kinase inhibitor or withdrawal of TGFbeta for 6-12 days. Interestingly, both cell cycle arresting/proapoptotic (Smads, p38 kinase) and antiapoptotic, proliferation and EMT-promoting signalling pathways (PI3K-PKB/Akt, ERK) were co-suppressed to low, but significant levels. Except for PI3K-Akt, TGFbeta-dependent downregulation of these signalling pathways in transdifferentiated (TD) cells was fully reversed upon TGFbeta withdrawal, together with partial re-induction of proliferation arrest and apoptosis. Co-injection of non-tumorigenic NMuMG cells with tumour-forming CHO cells oversecreting exogenous TGFbeta1 (CHO-TGFbeta1) allowed outgrowth of epithelioid cells in CHO-TGFbeta1 cell-induced tumours. These epithelial islands enhanced CHO-TGFbeta1 tumour cell proliferation, possibly due to chemokines (for example, JE/MCP-1) secreted by NMuMG/TD cells. We conclude that suppression of antiproliferative, proapoptotic TGFbeta signalling in TD cells may permit TGFbeta-dependent proliferation, survival and EMT-enhancing signalling pathways to act at low levels. Thus, TGFbeta may modulate its own signalling to facilitate switching from tumour suppression to tumour progression.

Smad2 suppresses the growth of Mv1Lu cells subcutaneously inoculated in mice.

Smad2 and Smad3 are intracellular signal transduction proteins of importance in transforming growth factor-beta (TGFbeta)-mediated inhibition of epithelial cell proliferation. Inactivating mutations in the Smad2 and Smad3 genes have been found in various human malignancies. Here, we show that expression of Smad2 leads to the inhibition of growth of Mv1Lu cells inoculated with Matrigel subcutaneously (s.c.) in severe combined immunodeficient (SCID) mice. In histological appearance, the Matrigel plugs with Smad2-transfected cells showed strongly reduced cell density, proliferation and angiogenesis compared with the small tumour nodules of similar size formed by the vector- or Smad3-transfected cells. The histological appearance of vector- and Smad3-transfected cells inoculated in mice was identical. Overexpression of Smad2 and Smad3 in Mv1Lu cells led to the inhibition of cell growth in three-dimensional cultures when compared with vector-transfected cells. Overexpression of Smad2 and Smad3 also decreased the hyperphosphorylation of pRb in Smad-transfected cells. Thus, increased expression of Smad2 leads to inhibition of Mv1Lu cell proliferation and a reduction in the growth of the Smad2-expressing cells inoculated in mice.

Growth inhibition of dermatofibrosarcoma protuberans tumors by the platelet-derived growth factor receptor antagonist STI571 through induction of apoptosis.

Dermatofibrosarcoma protuberans (DFSP) and giant cell fibroblastoma (GCF) are recurrent, infiltrative skin tumors that presently are treated with surgery. DFSP and GCF tumors are genetically characterized by chromosomal rearrangements fusing the collagen type Ialpha1 (COLIA1) gene to the platelet-derived growth factor B-chain (PDGFB) gene. It has been shown that the resulting COL1A1/PDGF-B fusion protein is processed to mature PDGF-BB. Autocrine PDGF receptor stimulation has therefore been predicted to contribute to DFSP and GCF tumor development and growth. Here we demonstrate presence of activated PDGF receptors in primary cultures derived from six different DFSP and GCF tumors. Three of the primary cultures were further characterized; their in vitro growth displayed an increased sensitivity to treatment with the PDGF receptor tyrosine kinase inhibitor STI571, as compared with normal fibroblasts. Transplantable tumors, displaying a DFSP-like histology, were established from one of the DFSP primary cultures. Treatment of tumor-bearing severe combined immunodeficient mice with STI571 reduced tumor growth. The growth-inhibitory effects in vitro and in vivo occurred predominantly through induction of tumor cell apoptosis. Our study demonstrates growth-inhibitory effects of PDGF receptor antagonists on human DFSP- and GCF-derived tumor cells and demonstrates that autocrine PDGF receptor stimulation provides antiapoptotic signals contributing to the growth of these cells. These findings suggest targeting of PDGF receptors as a novel treatment strategy for DFSP and GCF.

Antiangiogenic effects of latent antithrombin through perturbed cell-matrix interactions and apoptosis of endothelial cells.

Antithrombin is a plasma protein of the serpin superfamily that may occur as several conformational variants. The native form of antithrombin is a major regulator of blood clotting. In the present study, we have identified the mechanism underlying the antiangiogenic action of a heat-denatured form, denoted latent antithrombin. Fibroblast growth factor (FGF)-induced angiogenesis in the chick embryo and angiogenesis in mouse fibrosarcoma tumors were inhibited by treatment with latent antithrombin at 1 mg/kg/day. Thermolysin-cleaved and native antithrombin were less efficient in these respects. Treatment with latent antithrombin induced apoptosis of cultured endothelial cells and inhibited cell migration toward FGF-2. Under these conditions, FGF-2-stimulated FGF receptor kinase activity was unaffected. However, actin reorganization, activation of focal adhesion kinase, and focal adhesion formation were disturbed by latent antithrombin treatment of FGF-2-stimulated endothelial cells. These data indicate that latent antithrombin induces apoptosis of endothelial cells by disrupting cell-matrix interactions through uncoupling of focal adhesion kinase.

Predimerization of recombinant platelet-derived growth factor receptor extracellular domains increases antagonistic potency.

Platelet-derived growth factor (PDGF) is a dimeric growth factor acting through tyrosine kinase alpha- and beta-receptors. In both receptors, the extracellular parts are composed of five Ig-like domains. Functional mapping of the extracellular part of the receptors have shown that ligand-binding occurs to Ig-like domains 2 and 3 and that Ig-like domain 4 is involved in receptor-receptor interactions. Recombinant GST-fusion proteins of PDGF alpha-receptor Ig-like domains 1-4 and beta-receptor Ig-like domains 1-3 (alphaRD1-4-GST and betaRD1-3-GST) were generated and compared with their cleaved counterparts (alphaRD1-4 and betaRD1-3) with regard to their ability to block PDGF binding to cell surface receptors. In the case of both the alpha- and the beta-receptors, 100-1000-fold lower concentrations of the GST-fusion proteins were required, as compared to the cleaved forms, for inhibition of PDGF binding to cell surface receptors. alphaRD1-4-GST and betaRD1-3-GST, in contrast to alphaRD1-4 and betaRD1-3, were shown to occur as ligand independent dimers. Covalently cross-linked alphaRD1-4 dimers displayed a 50-fold increased potency as compared to alphaRD1-4. We thus conclude that the dimeric nature of alphaRD1-4-GST and betaRD1-3-GST is responsible for the high antagonistic potency of the fusion proteins.

Characterization of the chronic myelomonocytic leukemia associated TEL-PDGF beta R fusion protein.

The t(5;12) translocation, associated with chronic myelomonocytic leukemia, generates a novel gene encoding a protein, TEL-PDGF beta R, composed of the 154 amino-terminal amino acids of the transcription factor TEL and the transmembrane and intracellular part of the PDGF beta-receptor (PDGF beta R). TEL also occurs as a tumor-associated fusion partner for the tyrosine kinases c-ABL, JAK2 and TRK-C. Previous studies have demonstrated growth promoting activity of TEL-PDGF beta R and also indicated that the TEL moiety activates the tyrosine kinase of the PDGF beta R through the formation of TEL-PDGF beta R oligomers. We demonstrate that tyrosine phosphorylation of the fusion protein can be attenuated through overexpression of the TEL part of TEL-PDGF beta R, suggesting a strategy for antagonizing the signaling of TEL-PDGF beta R, and other TEL-fusion proteins containing tyrosine kinase domains. Comparison of BaF/3 cell lines expressing TEL-PDGF beta R and ligand-stimulated PDGF beta R revealed that only TEL-PDGF beta R expression conferred IL-3-independent growth, suggesting differences in signaling capacity of the two proteins. Finally, tyrosine residues 17 and 27 in TEL-PDGF beta R was identified as autophosphorylation sites in TEL-PDGF beta R.

The dermatofibrosarcoma protuberans-associated collagen type Ialpha1/platelet-derived growth factor (PDGF) B-chain fusion gene generates a transforming protein that is processed to functional PDGF-BB.

Dermatofibrosarcoma protuberans (DFSP) displays chromosomal rearrangements involving chromosome 17 and 22, which fuse the collagen type Ialpha1 (COLIA1) gene to the platelet-derived growth factor (PDGF) B-chain (PDGFB) gene. To characterize the functional and structural properties of the COLIA1/PDGFB fusion protein, we generated a stable NIH3T3 cell line that contained a tumor-derived chimeric gene resulting from a COIA1 intron 7-PDGFB intron 1 fusion. Expression of the fusion protein led to morphological transformation and increased growth rate of these cells. The PDGF receptor kinase inhibitor CGP57148B reversed the transformed phenotype and reduced the growth rate of COLIA1/PDGFB-expressing cells but had no effects on control cells. The presence of dimeric COLIA1/PDGFB precursors was demonstrated through PDGFB immunoprecipitations of metabolically labeled cells and also by PDGFB immunoprecipitations followed by immunoblotting with COLIA1 antibodies. Pulse-chase studies demonstrated that the COLIA1/PDGFB precursor was processed to an end product that was indistinguishable from wild-type PDGF-BB. Finally, COLIA1/PDGFB-expressing cells generated tumors after s.c. injection into nude mice, and tumor growth was reduced by treatment with CGP57148B. We conclude that the COLIA1/PDGFB fusion associated with DFSP contributes to tumor development through ectopic production of PDGF-BB and the formation of an autocrine loop. Our findings, thus, suggest that PDGF receptors could be a target for pharmacological treatment of DFSP and giant cell fibroblastoma, e.g., through the use of PDGF receptor kinase inhibitors such as CGP57148B.

Joint effect of shift work and adverse life-style factors on the risk of coronary heart disease.

OBJECTIVES: The joint effect of shift work and certain adverse life-style factors on coronary heart disease (CHD) was studied. METHODS: Base-line measurements were obtained for a 6-year follow-up of an industrially employed cohort (N= 1806), whose shiftwork status was recorded from a questionnaire filled out by a sample of the cohort. The CHD end points (codes 410-414 of the 9th revision of the International Classification of Diseases) were obtained from official Finnish registers. In order that the joint effects of shift work and life-style factors on the risk of CHD could be studied, dichotomized variables and their combinations as a dummy variable system in Cox's proportional hazards models were used. RESULTS: The relative risks were 1, 1.6[95% confidence interval (95% CI) 1.1-2.5], 1.3(95% CI 0.9-2.1), and 2.7(95% CI 1.8-4.1) for the following combinations of shift work (SW) and smoking (SM): SW-&SM-, SW-&SM+, SW+&SM-, and SW+&SM+, respectively; and the corresponding figures for shift work and obesity (BMI > or =28 kg/m2) were 1, 1.2(95% CI0.8-1.9), 1.3(95% CI0.9-1.9), and 2.3(95% CI1.5-3.6), respectively. In both cases the effect was at least multiplicative. For the shift workers the relative risk for CHD rose gradually with increasing numbers of adverse life-style factors, but for the day workers there was no clear dose-response pattern. CONCLUSIONS: Shift work seems to trigger the effect of other, lifestyle-related risk factors of CHD and therefore calls for active prevention among shift workers.

Combined effects of shift work and life-style on the prevalence of insomnia, sleep deprivation and daytime sleepiness.

OBJECTIVES: The combined effects of age, leisure-time physical activity, smoking, alcohol consumption, and different forms of shift work on the prevalence of sleep complaints and daytime sleepiness were studied among workers in industry, transport, and traffic. METHODS: Altogether 3020 subjects were studied using a psychosocial questionnaire. The participants were currently employed men, aged 45-60 years, from a postal and telecommunication agency, the railway company, and 5 industrial companies. On the basis of a factor analysis of an 11-item sleep questionnaire, the sleep complaints were grouped into the categories of insomnia, sleep deprivation, daytime sleepiness, and snoring. The importance of the shift schedule, age, and life-style factors as simultaneous predictors of the complaints was studied in a logistic regression analysis and an analysis of covariance. RESULTS: The prevalence of insomnia, sleep deprivation, and daytime sleepiness depended significantly on the shift system. All sleep complaints were more common in 2- and 3-shift work and in irregular shift work than in day work. The prevalence of daytime sleepiness was 20-37%, depending on the shift system. Leisure-time physical activity and alcohol consumption were the most important life-style factors predicting all sleep complaints, except snoring. The effects of physical activity and alcohol consumption differed for different shift schedules. CONCLUSIONS: Different shift systems, also 2-shift work and permanent night work, seem to increase the frequency of sleep complaints. Especially 3-shift work seems to interact with life-style factors by increasing the adverse effects and decreasing the beneficial effects on sleep and sleepiness.

Apoptotic response of spermatogenic cells to the germ cell mutagens etoposide, adriamycin, and diepoxybutane.

In testis, apoptosis is a way to eliminate damaged germ cells during their development. In this study, we evaluated the ability of three germ cell mutagens to induce apoptosis (or programmed cell death) at specific stages of rat seminiferous epithelial cycle. These chemicals include the cancer chemotherapy drugs etoposide and adriamycin and the butadiene metabolite diepoxybutane. According to our results, etoposide is a very potent inducer of apoptosis in male rat germ cells and the cell types most sensitive to it include all types of spermatogonia, zygotene, and early pachytene spermatocytes and meiotically dividing spermatocytes. Also, adriamycin causes an increase in apoptosis at specific stages of seminiferous epithelial cycle and the most sensitive cell types are type A3-4 spermatogonia, preleptotene, zygotene, and early pachytene spermatocytes. Diepoxybutane does not cause any significant increase in the frequency of apoptosis in rat testis. In addition, we studied whether p53 is taking part in the apoptotic response of spermatogenic cells by studying the levels of p53 protein in testis before and after chemical treatment. No accumulation of p53 in testis was seen after treatment with these three chemicals. The expression of two p53-regulated genes, p21WAF1 and mdm2, was also studied but no increase in the levels of mRNA of these genes was observed after treatment. The results indicate that apoptosis should be taken into consideration when the genotoxic effects of chemicals are evaluated in germ cells.

Shift work, occupation and coronary heart disease over 6 years of follow-up in the Helsinki Heart Study.

OBJECTIVES: The risk of coronary heart disease (CHD) in shift work and the possible pathways for CHD in industrial workers were studied along with the importance of shift work as an occupational class gradient of CHD risk. METHODS: Data from a psychosocial questionnaire and on life-style factors, blood pressure, and serum lipid levels were used for a follow-up study of a cohort of 1806 workers. CHD was determined from official Finnish registers. Cox's proportional hazards models were used with different covariates to evaluate the relative risks associated with shift work. RESULTS: All the blue-collar workers smoked more and a had higher systolic blood pressure than the white-collar workers. Three-shift workers scored low for job-decision latitude on the Karasek job stress scales. There were no differences in the total cholesterol or high-density lipoprotein cholesterol levels. When all the shift workers were compared with all the day workers, the relative risk of CHD was 1.5 [95% confidence interval (95% CI) 1.1-2.1] when only age was adjusted for and 1.4 (95% CI 1.0-1.9) when life-style factors, blood pressure, and serum lipids were also adjusted for. The blue-collar day workers and 2-shift and 3-shift workers had relative risks of 1.3 (95% CI 0.8-2.0), 1.9 (95% CI 1.1-3.4), and 1.7 (95% CI 1.1-2.7), respectively, when compared with the white-collar day workers. CONCLUSIONS: Shift work is an important part of the occupational gradient in CHD risk among industrial workers; some evidence was found for the hypothesis that a direct stress-related mechanism explains part of the increased CHD risk.

Respiratory symptoms and dermatoses among grinders and brazers of hard metal and stellite blades.

The objectives of the study were to determine whether grinders and brazers of hard metal and stellite blades have more respiratory symptoms and dermatoses than referents and to obtain information on the relation between respiratory symptoms and combined exposure to cobalt and wood dust. Two groups of workers exposed to cobalt (108 workers in the manufacture or maintenance of tools and 116 saw filers in the mechanical wood-processing industry) and two reference groups (106 rolling mill and 103 sawmill workers) were interviewed. The prevalence of ODTS-like symptoms (work-related cough, dyspnoea, or fever or chills) was higher for the saw filers than the sawmill referents. After adjustment for age, time spent in present work, smoking and atopy, saw filers had a higher risk for fever or chills than the other study groups. When the cobalt-exposed and unexposed workers were compared by smoking, differences in the prevalence of ODTS-like symptoms were found only for the non-smokers. The cobalt-exposed workers did not have a higher risk of hand dermatoses or symptoms of metal allergy than the unexposed workers. It seems that combined cobalt and wood dust exposure is associated with ODTS-like symptoms, especially among non-smoking workers.

Germ cell mutagenicity of three metabolites of 1,3-butadiene in the rat: induction of spermatid micronuclei by butadiene mono-, di-, and diolepoxides in vivo.

Three metabolites of the industrial chemical 1,3-butadiene (BD), namely butadiene monoepoxide (BMO, 3,4-epoxy-1-butene), diepoxide (DEB, 1,2;3,4-diepoxybutane), and diolepoxide (DE, 3,4- epoxybutane-1,2-diol) were studied for germ cell mutagenicity using the rat spermatid micronucleus (MN) test. All three epoxides increased slightly, but significantly, the frequency of spermatid MN. The most sensitive stage to the action of BMO and DEB was preleptotene (meiotic S phase) harvested at 18-day time intervals after treatment. The dose-response for BMO followed a second order curve at this time interval, with maximum MN induction at the dose of 186 mumol/kg and lower induction of higher doses. Late stages of the meiotic prophase (late pachytene-diplotene-diakinesis) also showed some sensitivity to the three epoxides. Stem cell spermatogonia were affected by DEB as observed by a slight induction of spermatid micronuclei 50 days after treatment. No clear cytotoxic effects were observed by measuring testicular weight or cell numbers of seminiferous epithelial stage 1 18 days after the treatments. DEB at the dose 387 mumol/kg caused a slight inhibition of spermatogonial DNA synthesis in stage I and a delay of meiotic DNA replication observed in stage XII 72 hr after treatment. Since BMO is able to induce spermatid MN in the rat, the present results, together with previous data, indicate that rat bone marrow MN results that are negative for both BD and BMO cannot directly predict mutagenicity in male germ cells. The results also emphasize that tissue; species, and strain-specific differences in metabolism have to be taken into account when the genetic risks of human butadiene exposure are evaluated. The results support the conclusion that 1,3-butadiene is a germ cell mutagen-possibly also in humans.

Role of deoxyribonucleic acid polymerase epsilon in spermatogenesis in mice.

Previous studies on DNA polymerase epsilon indicate that this enzyme is involved in replication of chromosomal DNA. In this study, we examined the expression of DNA polymerases alpha, delta, and epsilon during mouse testis development and germ cell differentiation. The steady-state levels of mRNAs encoding DNA polymerase epsilon and the recombination enzyme Rad51 remained constant during testis development, whereas the mRNA levels of DNA polymerases alpha and delta declined from birth until sexual maturity. Immunohistochemical staining methods, using a stage-specific model of the seminiferous epithelium, revealed dramatic differences between DNA polymerase alpha and epsilon distribution. As expected, DNA polymerase alpha and proliferating cell nuclear antigen showed relatively strong immunostaining in mitotically proliferating spermatogonia and even stronger staining in preleptotene cells undergoing meiotic DNA replication. The distribution of Rad51 was similar, but there was a dramatic peak in late pachytene cells. In contrast, DNA polymerase epsilon was detectable in mitotically proliferating spermatogonia but not in the early stages of meiotic prophase. However, DNA polymerase epsilon reappeared in late pachytene cells and remained through the two meiotic divisions, and was present in haploid spermatids up to the stage at which the flagellum starts developing. Overall, the results suggest that DNA polymerase epsilon functions in mitotic replication, in the completion of recombination in late pachytene cells, and in repair of DNA damage in round spermatids. In contrast, DNA polymerases alpha and delta appear to be involved in meiotic DNA synthesis, which occurs early in meiotic prophase, in addition to functioning in DNA replication in proliferating spermatogonia.

Micronuclei are induced in rat spermatids in vitro by 1,2,3,4-diepoxybutane but not by 1,2-epoxy-3-butene and 1,2-dihydroxy-3,4-epoxybutane.

The genotoxic effects of three 1,3-butadiene metabolites, 1,2-epoxy-3-butene (monoepoxide, EB), 1,2,3,4-diepoxybutane (diepoxide, DEB) and 1,2-dihydroxy-3,4-epoxybutane (diolepoxide, DiolEB), on male rat germ cells were studied by the meiotic micronucleus method in vitro. Seminiferous tubular segments from stages XII to XIII containing late pachytene-diakinetic spermatocytes were cultivated in the presence of the test chemical for 4 days. During the culture, spermatocytes passed through meiotic divisions and developed into early spermatids in which micronuclei could be scored. DEB was found to be a very potent micronucleus inducer in rat meiosis. All concentrations tested (5-20 microM) were able to cause a statistically significantly higher frequency of micronuclei (P < 0.05) compared with controls and a linear dose-dependent trend for micronucleus induction was seen (P < 0.01). However, EB and DiolEB caused no increase in micronucleus frequencies in spermatids at the concentrations tested (100-1000 microM for EB and 10-100 microM for DiolEB) and at higher concentrations cytotoxic effects were seen upon dividing cells causing a significant reduction in the number of spermatids. According to these results DEB is the most genotoxic butadiene metabolite in rat germ cells during meiosis.

Expression of p53 in normal and gamma-irradiated rat testis suggests a role for p53 in meiotic recombination and repair.

In testis, the expression of tumor suppressor protein p53 is stronger than in other tissues suggesting a role for it in spermatogenesis. We have studied the expression of p53 in both unirradiated and gamma-irradiated rat testis using the stage-specific model of rat seminiferous epithelium. Our results show that p53 is expressed during meiosis in normal rat spermatogenesis and its expression is localized to the preleptotene-early pachytene spermatocytes. The most prominent expression is in zygotene - early pachytene spermatocytes (stages XIII-I of seminiferous epithelium). After irradiation p53 levels increased in a time and a dose-dependent manner being highest with the doses of 6.0 and 12.0 Gy and 4 h after irradiation. This increase occurs in the same cells that normally express elevated levels of p53. These results support the view that p53 is involved in meiosis of the male rat and we suggest that p53 has a role in recombinational processes and/or formation of the synaptonemal complex. We also demonstrate that p53 takes part in the response of primary spermatocytes to irradiation gamma-induced DNA damage.

Stage-specific DNA synthesis of rat spermatogenesis as an indicator of genotoxic effects of vinblastine, mitomycin C and ionizing radiation on rat spermatogonia and spermatocytes.

We have studied the effects of three known mutagens: vinblastine sulphate, mitomycin C and local irradiation of testes on the stage-specific DNA synthesis in the rat testis by using transillumination assisted microdissection of rat seminiferous tubules. It enables us to investigate the sensitivity of different types of spermatogonia and preleptotene spermatocytes to the genotoxic effects of these agents. According to our results, spermatogonia and preleptotene spermatocytes are quite resistant to the action of vinblastine at the treatment times and the doses used. After treatment with mitomycin C, type A2, A3 and A4 spermatogonia seem to be the first cell types affected, which shows itself as a reduction in the DNA synthesis at stages I, II-III, XIII-XIV of the epithelial cycle two and/or three days after the treatment. It also seems that they are mostly affected during the S-phase of their cell cycles. In addition, preleptotene spermatocytes are also sensitive to the action of mitomycin C when they are treated in the G1 phase of the cell cycle. The local irradiation of 3 Gy has severe effects on the spermatogonia of rat testis which can be seen already 18 h after the treatment and becomes more evident 42 and 66 h after the treatment as a reduction of DNA synthesis at stages XII-V. Type A spermatogonia (A1-A4) seem to be the most sensitive cell types to the action of irradiation. This study indicates that the novel method of stage-specific DNA synthesis in rat spermatogenesis allows detailed studies of sensitivities in differentiating spermatogonia to genotoxic agents.

Effects of vinblastine and colchicine on male rat meiosis in vivo: disturbances in spindle dynamics causing micronuclei and metaphase arrest.

We have studied the effects of vinblastine sulfate (VBL) and colchicine (COL) on male rat in vivo and in vitro meiosis. A novel methodology based on isolating a segment of seminiferous tubules containing meiotically dividing spermatocytes was applied. During meiotic divisions at stage XIV of rat spermatogenesis, both chemicals induced only low frequencies of micronuclei (MN), 0.8-3.2 MN/1,000 spermatids. Fluorescence in situ hybridization experiments in mice with the mouse centromere-specific gamma-satellite DNA probe showed that 50.7% of VBL-induced MN and 56.6% of COL-induced MN were centromere positive, indicating that the MN induced by both chemicals contained detached chromosomes. The inhibition of cell proliferation was determined by counting the number of cells arrested at metaphase during the first meiotic (MI) or the second meiotic (MII) division. VBL was found to be a potent inducer of cell death while COL was not. The direct effects of VBL and COL on the meiotic spindles were evaluated using immunohistochemistry with anti-alpha-tubulin and confocal microscopy. In the control animals a significant difference was observed between the mean length of metaphase spindles of MI and MII. Both were dramatically decreased 6 hr after treatment with 2.0 mg/kg of VBL and 0.8 mg/kg of COL, respectively. At 18 hr after COL injection the spindles had about the same length as in the controls. However, the VBL-induced shortening was even more evident at 18 hr for both MI and MII. The possible reasons for observed differences between the two chemicals and between meiosis and mitosis are discussed.