Spontaneous death of rat chloroleukaemia cells induced by an endogenous growth inhibitor.

OBJECTIVES: When rat chloroleukaemia (CHL) cells are grown undisturbed in a confined space, a genomic long interspersed nuclear element (LINE) is transcriptionally activated at a relatively low population density, followed by the retrotransposition of LINE and population death. This death programme is fundamentally different from conventional cell death pathways. MATERIALS AND METHODS: This work is essentially based on the re-analysis of relevant, old experimental data. Elemental analysis of a highly purified, long-stored inhibitor sample was performed. Genomic sequence searches were performed using the basic local alignment search tool (BLAST). RESULTS: This death programme is initiated by an endogenous inhibitor secreted by CHL cells. The inhibitor is almost certainly identical to the pentapeptide pyroGlu-Glu-Asp-Cys-Lys, shown to be a cell line-specific inhibitor of normal granulocytic cells. The inhibitor is derived from a highly conserved short open reading frame in mammalian genomes. CONCLUSIONS: Although spontaneous population death may be a biological oddity restricted to rat CHL cells, we suggest that this death programme is responsible for the eradication of cancer cells following treatment with an inhibitor administered exogenously.

Effects of 50 Hz magnetic field on cell cycle kinetics and the colony forming ability of budding yeast exposed to ultraviolet radiation.

To investigate the effects of extremely low frequency magnetic fields on ultraviolet radiation (UV) exposed budding yeast, haploid yeast (Saccharomyces cerevisiae) cells of the strain SEy2101a were exposed to 50 Hz sine wave magnetic field (MF) of 120 microT with simultaneous exposure to UV radiation. Most of the UV energy was in the UVB range (280-320 nm). The biologically weighted (CIE action spectrum) dose level for the UV radiation was 175 J/m2. We examined whether 50 Hz MF affected the ability of UV irradiated yeast cells to form colonies (Colony Forming Units, CFUs). In addition, the effect of coexposure on cell cycle kinetics was investigated. Although the significant effect of MF on the cell cycle phases of UV exposed yeast cells was seen only at one time point, the overall results showed that MF exposure may influence the cell cycle kinetics at the first cycle after UV irradiation. The effect of our particular MF exposure on the colony forming ability of the UV irradiated yeast cells was statistically significant 420 min after UV irradiation. Moreover, at 240, 360, and 420 min after UV irradiation, there were fewer CFUs in every experiment in (UV+MF) exposed populations than in only UV exposed yeast populations. These results could indicate that MF exposure in conjunction with UV may have some effects on yeast cell survival or growth.

Transforming growth factor beta receptor induction in herniated intervertebral disc tissue: an immunohistochemical study.

Transforming growth factor beta (TGF-beta) is a potent inducer of angiogenesis and fibrogenesis. There is presently little information about the pathophysiological function of TGF-beta in herniated disc tissue. In order to analyze the cellular role and activation of TGF-beta after disc herniation we immunostained frozen material from 38 disc herniation operations and from eight macroscopically normal discs from organ donors. Polyclonal TGF-beta-I, TGF-beta-II and TGF-beta receptor type II antibodies were used with the avidin biotin complex (ABC-) immunoperoxidase method. All the herniated discs were TGF-beta immunopositive. Such immunoreactivity was mainly associated with disc cells. In a few samples, capillaries were also TGF-beta immunopositive. Immunopositivity was similarly observed in the control discs. To analyze possible differences between the two groups, we calculated the ratio of immunopositive disc cells. For all three antibodies, a statistically significantly (Mann-Whitney test, P = 0.0001) higher number of disc cells showed immunopositivity in the herniated discs. The increase in TGF-beta receptor immunopositivity suggested induction of TGF-beta receptors in herniated discs. Our results support an active regulatory role for TGF-beta in disc cell metabolism.

Chernobyl fallout and outcome of pregnancy in Finland.

Possible effects of Chernobyl fallout on outcome of pregnancy in Finland were evaluated in a nationwide follow-up study. The outcomes were the rate of live births and stillbirths, pregnancy loss, and induced abortions by municipality. Exposure was assessed based on nationwide surveys of radiation dose rate from the Chernobyl fallout, from both external and internal exposures. Using these measurements, we estimated the monthly dose rate for each of the 455 Finnish municipalities. On average, the dose rate from Chernobyl fallout reached 50 microSv per month in May 1986--a doubling of the natural background radiation. In the most heavily affected area, 4 times the normal background dose rates were recorded. Given the underlying regional differences in live birth, stillbirth, and abortion rates, we used longitudinal analysis comparing changes over time within municipalities. A temporary decline in the live birth rate had already begun before 1986, with no clear relationship to the level of fallout. A statistically significant increase in spontaneous abortions with dose of radiation was observed. No marked changes in induced abortions or stillbirths were observed. The decrease in the live birth rate is probably not a biological effect of radiation, but more likely related to public concerns of the fallout. The effect on spontaneous abortions should be interpreted with caution, because of potential bias or confounding. Further, there is little support in the epidemiologic literature on effects of very low doses of radiation on pregnancy outcome.

p53 and K-ras gene mutations in carcinoma of the rectum among Finnish women.

AIMS/BACKGROUND: The aim of this study was to identify p53 and K-ras gene mutations in carcinoma of the rectum among Finnish women. Mutation patterns might give clues to aetiological factors when comparisons are made with other human tumours. METHODS: Of 134 women with carcinoma of the rectum, paraffin wax embedded specimens of the tumour tissue were obtained from 118 patients. Genomic DNA was extracted, and exons 4-8 of the p53 gene and codons 12/13 and 61 of the K-ras gene were amplified, and analysed for mutations by single strand conformation polymorphism and direct sequencing. The production of p53 and K-ras proteins was studied by immunohistochemistry. RESULTS: The overall crude frequency for mutations in the p53 gene was 35% but the true frequency appears to be higher (up to 56%). In the K-ras gene, the mutation frequency (15%) was significantly lower than that reported for colon cancer. In the p53 gene, the mutation frequency increased significantly with patient age. In a high proportion of patients (14%) the rectal tumours contained small subclones of tumour cells that displayed extremely rare mutations at codons 110 and 232 of the p53 gene. Hot spot codon 175 mutations were significantly less common in rectal cancer than in cancer of the colon. CONCLUSIONS: Rectal cancer among Finnish women has characteristics in the mutations of the p53 and K-ras genes that are uncommon in other human tumours, including cancer of the colon. A biological explanation of these findings is not clear at present, but might be associated with an unidentified genetic factor in Finland.

Possible cocarcinogenic effects of ELF electromagnetic fields may require repeated long-term interaction with known carcinogenic factors.

Literature on cancer-related biological effects of extremely low frequency (ELF) magnetic fields (MF) is discussed in the light of the current understanding of carcinogenesis as a multistep process of accumulating mutations. Different animal models and study designs have been used to address possible cocarcinogenic effects of MFs. Based on a comparison of the results, we propose a hypothesis that MF exposure may potentiate the effects of known carcinogens only when both exposures are chronic. We also discuss possible mechanisms of MF effects on carcinogenesis and the adequacy of the classical two-step initiation/promotion animal experiments for simulating human exposure to the complex mixture of environmental carcinogens. We conclude that experiments designed according to the two-step concept may not be sufficient for studying the possible role of MF in carcinogenesis. Possible further animal studies are more likely to be productive if they include models that combine chronic exposure to MFs with long-term exposures to known carcinogens.

p53 gene mutations in neoplastic transformation of C3H 10T1/2 and severe combined immunodeficiency fibroblasts.

The relevance of p53 mutations to the neoplastic malignant transformation of rodent fibroblasts by genotoxic physical and chemical agents is not clear. In the present study, we investigated p53 mutations (in exons 5-8) in non-transformed and neoplastically transformed C3H 10T1/2 and severe combined immunodeficiency (SCID) cells. No p53 mutations were detected in 15 neoplastically transformed (two spontaneous, one 3-methylcholanthrene-induced, seven gamma-ray-induced and five 'hot particle'-induced) and two non-transformed 10T1/2 cells. Wild-type p53 gene was also detected in all non-transformed (immortalized) SCID cell lines analyzed (four lines) whereas all three neoplastically transformed (two spontaneous, one gamma-ray-induced) cell lines displayed missense mutations in the p53 gene. These mutations were all transitions: A > G in codon 123, G > A in codon 152, and C > T in codon 238. We conclude that mutation in the p53 gene appears to be an infrequent event in 10T1/2 cells regardless of the transforming agent, but a frequent event in the neoplastic transformation of immortalized SCID cells. Non-transformed SCID cells are deficient in repair of DNA double-strand breaks, and neoplastically transformed cells are assumed to be deficient as well.

Association of p53, K-ras and proliferating cell nuclear antigen with rat lung lesions following exposure to simulated nuclear fuel particles.

Expression of p53, K-ras, and proliferating cell nuclear antigen (PCNA) and mutations of p53 and K-ras genes in lung lesions of Han/Wistar rats were investigated by immunohistochemistry and direct DNA sequencing following a long-term exposure of animals to neutron-activated UO2 particles. The p53 protein was overexpressed in all five malignant tumors, in 62% of benign tumors, and in 42% of hyperplastic lesions examined. K-ras protein and PCNA levels were only slightly elevated in all types of lung lesions. In three malignant tumors a C-->T transition was detected in codon 288 (human 290) of the p53 gene, but this mutation was not present in seven other tumors analyzed. No mutations were detected in codons 12/13 and 61 of the K-ras gene in any of the five tumors analyzed. Our findings suggest that K-ras overexpression is a rare alteration, whereas p53 protein overexpression (sometimes associated with mutated p53 gene), as assessed with the CM5 antibody, is a relatively common phenomenon in hot particle-induced preneoplastic and neoplastic rat lung lesions.

K-ras and p53 mutations and overexpressions as prognostic factors in female rectal carcinoma.

BACKGROUND: The present study was undertaken to determine the prognostic significance of K-ras, p53 and bcl-2 in female rectal carcinoma. MATERIALS AND METHODS: The mutations in K-ras and p53 genes were analysed using SSCP and direct sequencing. The expression of K-ras, bcl-2 and p53 proteins was determined immunohistochemically. RESULTS: Mutations of K-ras and p53 genes were detected in 12% and 38% of the tumours, respectively. The prevalence of K-ras overexpression was 67%. K-ras mutations were not associated with survival. However, more favourable survival was observed for patients with K-ras overexpression than with normal expression (adjusted hazard ratio from Cox model 0.4, 95% CI 0.2-0.8). Mutation or overexpression of p53 were not associated with survival. CONCLUSIONS: It may be possible, that the mutations and protein overexpression of K-ras and p53 in female rectal carcinoma have different clinical impact on patient survival as suggested in previous studies concerning colorectal carcinoma of both sexes.

Effects of 50 Hz magnetic fields on UV-induced skin tumourigenesis in ODC-transgenic and non-transgenic mice.

PURPOSE: To study the possible role of 50 Hz magnetic fields (MF) in UV-induced skin tumourigenesis using a sensitive animal model. MATERIALS AND METHODS: Transgenic mice (line K2) over-expressing the human ornithine decarboxylase (ODC) gene and their non-transgenic littermates were exposed for 10.5 months to UV-only or a combination of UV and a continuous (100 microT) or an intermittent MF with varying intensity (1.3-130 microT). RESULTS: Both MF exposure and transgenicity enhanced the onset rate of macroscopically detectable tumours, but the effect was statistically significant only for the MF exposure (p < 0.015). The number of animals bearing malignant tumours was low and similar in all exposure groups. Epidermal cysts (EC) appeared to be strongly associated with both MF exposure and high ODC activity (transgenic animals). However, EC are not known to be associated with carcinogenesis. The UV-only or combined UV and MF exposure did not affect the ODC activities measured at the end of the exposure. CONCLUSIONS: These results support the proposed tumour-promoting effect of MF, but do not suggest an important role for increased ODC activity in this process.

The Estonian study of Chernobyl cleanup workers: I. Design and questionnaire data.

Nearly 2% of the male population of Estonia aged 20-39 years were sent to Chernobyl to assist in the cleanup activities after the reactor accident. A cohort of 4,833 cleanup workers was assembled based on multiple and independent sources of information. Information obtained from 3,704 responses to a detailed questionnaire indicated that 63% of the workers were sent to Chernobyl in 1986; 54% were of Estonian and 35% of Russian ethnicity; 72% were married, and 1,164 of their 5,392 children were conceived after the Chernobyl disaster. The workers were less educated than their counterparts in the general population of Estonia, and only 8.5% had attended university. Based on doses entered in worker records, the mean dose was 11 cGy, with only 1.4% over 25 cGy. Nearly 85% of the workers were sent as part of military training activities, and more than half spent in excess of 3 months in the Chernobyl area. Thirty-six percent of the workers reported having worked within the immediate vicinity of the accident site; 11.5% worked on the roofs near the damaged reactor, clearing the highly radioactive debris. The most commonly performed task was the removal and burial of topsoil (55% of the workers). Potassium iodide was given to over 18% of the men. The study design also incorporates biological indicators of exposure based on the glycophorin A mutational assay of red blood cells and chromosome translocation analyses of lymphocytes; record linkage with national cancer registry and mortality registry files to determine cancer incidence and cause-specific mortality; thyroid screening examinations with ultrasound and fine-needle biopsy; and cryopreserved white blood cells and plasma for future molecular studies. Comprehensive studies of Chernobyl cleanup workers have potential to provide new information about cancer risks due to protracted exposures to ionizing radiation.

The Estonian study of Chernobyl cleanup workers: II. Incidence of cancer and mortality.

A cohort of 4,742 men from Estonia who had participated in the cleanup activities in the Chernobyl area sometime between 1986 and 1991 and were followed through 1993 was analyzed with respect to the incidence of cancer and mortality. Incidence and mortality in the cleanup workers were assessed relative to national rates. No increases were found in all cancers (25 incident cases compared to 26.5 expected) or in leukemia (no cases observed, 1.0 expected). Incidence did not differ statistically significantly from expectation for any individual cancer site or type, though lung cancer and non-Hodgkin's lymphoma both occurred slightly more often than expected. A total of 144 deaths were observed [standardized mortality ratio (SMR) = 0.98; 95% confidence interval (CI) = 0.82-1.14] during an average of 6.5 years of follow-up. Twenty-eight deaths (19.4%) were suicides (SMR = 1.52; 95% CI = 1.01-2.19). Exposure to ionizing radiation while at Chernobyl has not caused a detectable increase in the incidence of cancer among cleanup workers from Estonia. At least for the short follow-up period, diseases directly attributable to radiation appear to be of relatively minor importance when compared with the substantial excess of deaths due to suicide.

Biodosimetry of Chernobyl cleanup workers from Estonia and Latvia using the glycophorin A in vivo somatic cell mutation assay.

The reactor accident at Chernobyl in 1986 necessitated a massive environmental cleanup that involved over 600,000 workers from all 15 Republics of the former Soviet Union. To determine whether the whole-body radiation received by workers in the course of these decontamination activities resulted in a detectable biological response, over 1,500 blood samples were obtained from cleanup workers sent from two Baltic countries, Estonia and Latvia. Here we report the results of studies of biodosimetry using the glycophorin A (GPA) locus in vivo somatic cell mutation assay applied to 734 blood samples from these workers, to 51 control samples from unexposed Baltic populations and to 94 samples from historical U.S. controls. The data reveal inconsistent evidence that the protracted radiation exposures received by these workers resulted in a significant dose-associated increase in GPA locus mutations compared with the controls. Taken together, these data suggest that the average radiation exposure to these workers does not greatly exceed 10 cGy, the minimum levels at which radiation effects might be detectable by the assay. Although the protracted nature of the exposure may have reduced the efficiency of induction of GPA locus mutations, it is likely that the estimated physical doses for these cleanup worker populations (median reported dose 9.5 cGy) were too low to result in radiation damage to erythroid stem cells that can be detected reliably by this method.

The bcl-2 gene status of human head and neck cancer cell lines.

The bcl-2 oncogene was originally found in the translocation in a pre-B cell acute lymphocytic leukemia cell line. Since then a high expression of Bcl-2 has been found in many types of cancer. The bcl-2 gene encodes an intracellular membrane-associated protein. Overexpression of bcl-2 inhibits apoptosis induced by many drugs and radiation. In this study the bcl-2 gene status of 9 human head and neck squamous cell carcinoma cell lines was studied. Mutations of the bcl-2 gene were studied at mRNA and DNA levels. The presence and abundance of the Bcl-2 protein in cells were also investigated. In earlier studies the p53 tumour suppressor gene was screened for point mutations, and the radiosensitivity of these cell lines was measured. We were able to amplify bcl-2 cDNA from 5 of the 9 cell lines, which shows that bcl-2 was expressed in these cells. No point mutations were found in the bcl-2 gene in any of these cell lines. Loss of heterozygosity was observed in 2 cell lines at the bcl-2 locus, and these cell lines had no detectable levels of bcl-2 mRNA or Bcl-2 protein. The Bcl-2 protein was abundant in the cell lines with the wild-type p53 gene, and these cell lines were radioresistant. The Bcl-2 protein was also found in many other cell lines in mitotic cells. It seems that cells expressing bcl-2 are radioresistant, and even functional p53 cannot induce apoptosis in these cells.

p53 mutations in human head and neck cancer cell lines.

The p53 tumour suppressor gene is commonly mutated in human cancers. We performed a molecular analysis of the frequency and spectrum of p53 gene mutations in 40 cell lines (23 from oral cavity tumours and 17 from larynx tumours) derived from 33 patients with squamous cell carcinoma of the head and neck (SCCHN). Using PCR, SSCP, and sequence analysis, we detected the mutated p53 gene in 26 patients (79%); in 23 patients (70%) the wild-type allele of the p53 gene was deleted. Four patients had 2 p53 gene mutations each, and thus the total number of p53 mutations observed was 30. Seven patients had 2 cell lines each, established from the primary and recurrent/metastatic tumour, and the status of the p53 gene (mutant or normal) was identical in both cell lines. Forty percent of the mutations were transitions, 33% transversions, and 27% deletions, insertions and other more complicated changes. In oral cavity tumours the predominant mutation type was G:C-->A:T transition at a CpG site (50% of mutations), and in larynx tumours the predominant type was G:C-->T:A transversion (50% of mutations). These suggest endogenous and exogenous factors in tumour etiology. The G:C-->T:A transversions in larynx tumours are probably associated with mutagenic components in the cigarette smoke, but the causative factor in G:C-->A:T transitions (apparent oxidative damage) remains to be identified.

Platelet-derived growth factor and vascular endothelial growth factor expression in disc herniation tissue: and immunohistochemical study.

Angiogenesis is essential in tissue growth and regeneration. There are several factors that are able to stimulate vascular endothelial cell growth, including platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF). Disc herniation tissue (DHT) contains vascular ingrowth, which promotes granulation tissue formation. In this study we observed 50 disc herniations for PDGF and VEGF immunoreactivity. PDGF immunopositivity was detected in 38 samples (78%). In 28 samples (56%) there were PDGF immunopositive capillaries, PDGF immunopositive disc cells were detected in 19 samples (38%) and PDGF immunopositive fibroblasts in 6 DHT samples (12%). VEGF immunopositive capillaries were identified in 44 DHT samples (88%). For neither growth factor was immunopositivity dependent on preoperative radicular pain duration. In extrusions (n = 25) VEGF immunopositive capillaries were detected in 23 samples (92%) and PDGF immunopositivity in 21 samples (84%). PDGF immunopositivity was more commonly associated with capillaries than with nuclei of disc cells. In sequesters (n = 20) VEGF immunopositive capillaries were identified in all samples and PDGF immunopositivity in 16 (80%). As in extrusions, PDGF immunoreaction was more prevalent in capillaries than in disc cells. Patient age did not relate to VEGF expression. In all age groups it was higher than 80%. Thus capillaries in disc herniation tissue are evidently newly formed and our results demonstrate that PDGF and VEGF participate in the neovascularization process. The presence of PDGF in fibroblasts and in disc cells suggests that this growth factor regulates the function of these cells, possibly the proliferation of the cells and the production of extracellular matrix components.

Radon and lung cancer in Finland.

A study with 291 cases and 495 controls on indoor radon and lung cancer incidence was conducted in a Finnish population residing in a high-exposure area. Relative risks of 1.8 and 1.5 for the incidence of lung cancer were observed for those exposed to concentrations of 95-185 Bq m(-3) and 186 Bq m(-3), respectively. The increase in risk was not statistically significant.

Indoor radon exposure and risk of lung cancer: a nested case-control study in Finland.

BACKGROUND: Inhaled radon has been shown to cause lung cancer among underground miners exposed to very high radon concentrations, but the results regarding the effects of residential radon have been conflicting. PURPOSE: Our aim was to assess the effect of indoor radon exposure on the risk of lung cancer. METHODS: To investigate this effect, a nested case-control study was conducted in Finland. The subjects of the study were the 1973 lung cancer case patients (excluding patients with cancers of the pleura) diagnosed from January 1, 1986, until March 31, 1992, within a cohort of Finns residing in the same one-family house from January 1, 1967, or earlier, until the end of 1985 and 2885 control subjects identified from the same cohort and matched by age and sex. In September 1992, a letter was sent to all study subjects or proxy respondents explaining the purpose and methods of the study. After giving informed consent, the study participants were asked to fill out a questionnaire on smoking habits, occupational exposures, and other determinants of lung cancer risk and radon exposure. The odds ratio (OR) of lung cancer was estimated from matched and unmatched logistic regression analyses relative to indoor radon concentration assessed by use of a 12-month measurement with a passive alpha track detector. RESULTS. Five hundred seventeen case-control pairs were used in the matched analysis, and 1055 case subjects and 1544 control subjects were used in the unmatched analysis. The OR of lung cancer for indoor radon exposure obtained from matched analysis was 1.01 (95% confidence interval [CI] = 0.94-1.08) per 2.7 pCi/L (100 Bq m-3) after adjustment for the cigarette smoking status, intensity, duration, and age at commencement of smoking by subjects. For indoor radon concentrations 1.4-2.6, 2.7-5.3, 5.4-10.7, and 10.8-34.5 pCi/L (50-99, 100-199, 200-399, and 400-1277 Bq m-3, respectively), the matched ORs were 1.03 (95% CI = 0.84-1.26), 1.00 (95% CI = 0.78-1.29), 0.91 (95% CI = 0.61-1.35), and 1.15 (95% CI = 0.69-1.93), respectively, relative to the concentration below 1.4 pCi/L (0-49 Bq m-3). The unmatched analysis yielded similar results with somewhat smaller CIs. In the analyses stratified by age, sex, smoking status, or histologic type of lung cancer, no statistically significant indications of increased risk of lung cancer related to indoor radon concentration were observed for any of the subgroups. CONCLUSIONS: Our results do not indicate increased risk of lung cancer from indoor radon exposure. IMPLICATION: Indoor radon exposure does not appear to be an important cause of lung cancer.