In vitro model cell systems are important tools for studying mechanisms of radiation‐induced neoplastic transformation of human epithelial cells. In our study, the human thyroid epithelial cell line HTori‐3 was analyzed cytogenetically following exposure to different doses of α‐ and γ‐irradiation and subsequent tumor formation in athymic nude mice. Combining results from G‐banding, comparative genomic hybridization, and spectral karyotyping, chromosome abnormalities could be depicted in the parental line HTori‐3 and in nine different HTori lines established from the developed tumors. A number of chromosomal aberrations were found to be characteristic for simian virus 40 immortalization and/or radiation‐induced transformation of human thyroid epithelial cells. Common chromosomal changes in cell lines originating from different irradiation experiments were loss of 8q23 and 13cen‐q21 as well as gain of 1q32‐qter and 2q11.2‐q14.1. By comparison of chromosomal aberrations in cell lines exhibiting a different tumorigenic behavior, cytogenetic markers important for the tumorigenic process were studied. It appeared that deletions on chromosomes 9q32‐q34 and 7q21‐q31 as well as an increased copy number of chromosome 20 were important for the tumorigenic phenotype. A comparative breakpoint analysis of the marker chromosomes found and those observed in radiation‐induced childhood thyroid tumors from Belarus revealed a coincidence for a number of chromosome bands. Thus, the data support the usefulness of the established cell system as an in vitro model to study important steps during radiation‐induced malignant transformation in human thyroid cells. © 2001 Wiley‐Liss, Inc.

Telomere lengths in radiosensitive murine lymphoma cells L5178Y-S and parental radioresistant L5178Y cells were measured by quantitative fluorescence in situ hybridization. Results revealed a 7-fold reduction in telomere length in radiosensitive cells (7 kb) in comparison with radioresistant cells (48 kb). Therefore, it was reasoned that telomere length might be used as a marker for chromosomal radiosensitivity. In agreement with this hypothesis, a significant inverse correlation between telomere length and chromosomal radiosensitivity was observed in lymphocytes from 24 breast cancer patients and 5 normal individuals. In contrast, no chromosomal radiosensitivity was observed in mouse cell lines that showed shortened telomeres, possibly reflecting differences in radiation responses between primary cells and established cell lines. Telomere length abnormalities observed in radiosensitive cells suggest that these two phenotypes may be linked.

Drugs used mainly for the treatment of hypertension, such as angiotensin I-converting enzyme (ACE) inhibitors, can cause pancytopenia. The underlying cause of this side effect remains unknown. In the present study, long-term bone marrow cultures (LTBMCs) were utilized to evaluate the role of captopril (D-3-mercapto-2-methylpropionyl-L-proline), one of the potent ACE inhibitors, in regulating hematopoietic stem/progenitor cell proliferation. Captopril (10(-6) M final concentration) was added to LTBMCs at the beginning of the culture period and at weekly intervals for six weeks. There was no toxicity to the bone marrow cells as measured by the unchanged cell number in the nonadherent layer during the whole culture period, and there was an increased cellularity of the adherent layer at the end of the six weeks of treatment. However, captopril decreased the proportion of granulocyte-macrophage colony-forming cells (GM-CFCs) in S phase at weeks 2 and 3 as well as that of high proliferative potential colony-forming cells (HPP-CFCs) at week 3 in the nonadherent layer. There was no change in the kinetics of the GM-CFCs and HPP-CFCs present in the adherent layer. These results suggest that captopril causes myelosuppression by inhibiting hematopoietic cell proliferation of progenitor and stem cells rather than depleting cells of the bone marrow microenvironment.

The effect of Angiotensin I-converting enzyme (ACE) inhibitors on their own and in combination with the peptide AcSDKP on the proliferation of hematopoietic stem cells has been investigated. Hematopoietic stem cells from murine bone marrow induced into cell cycle following exposure to 2 Gy gamma-irradiation were incubated in vitro for up to 24 h in the presence of medium, captopril/lisinopril, AcSDKP, and AcSDKP with either ACE inhibitor, Hematopoietic stem cells were monitored using the high proliferative potential-colony forming cell-1 (HPP-CFC-1) population cloned in the presence of human IL-I beta, murine IL-3, and murine M-CSF. No significant inhibitory effect was observed in the presence of AcSDKP on its own and AcSDKP in combination with lisinopril, However, there was a significant inhibition of stem cell cycling when AcSDKP and captopril were combined, This suggests that captopril inhibits AcSDKP breakdown better than lisinopril, The combination of AcSDKP and captopril also had an inhibitory effect on cell recruitment into S phase, The fact that a combination of AcSDKP and captopril snitches cycling hematopoietic stem cells out of cycle indicates the importance of the N-active catalytic site of ACE in AcSDKP hydrolysis in vitro, Thus, AcSDKP in combination with appropriate ACE inhibitors may be of use in regulating the proliferation of hematopoietic stern cells in vitro.

The responses to X-rays of three simian virus 40 (SV40) immortalized but non-tumourigenic human bladder epithelial cell lines have been compared with a malignant bladder epithelial line using the micronucleus assay. A linear increase in induced micronuclei (MN) was observed for all four cell lines with increasing X-ray dose. The three SV40 immortalized lines were found to be significantly less sensitive than the malignant cell line. Spontaneous levels of MN indicate that certain cell lines within the SV40 immortalized lines have a higher genetic instability. These cell lines may have a predisposition towards the generation of a fully transformed phenotype when treated with carcinogenic agents.

Testosterone-induced DNA synthesis in cultured rat ventral prostate was used to compare the direct effects of Estracyt and Emcyt with that of their metabolite, estramustine, and their carrier-hormone, oestradiol-17 beta on prostatic growth. In serum-supplemented medium (5-20% FCS), all the compounds were equally effective in suppressing testosterone stimulated DNA synthesis which was reduced by between 40-50%, whereas in serum-free medium the estramustine compounds were consistently less effective than oestradiol-17 beta. In the presence of 4 X 10(-9) M testosterone in serum-free medium, stimulated DNA synthesis was reduced by 15-30% following incubation with 4 X 10(-7) M of Estracyt, Emcyt and estramustine and by 60% with 4 X 10(-7) M oestradiol-17 beta. Thus, none of the estramustine compounds appear to offer any selective advantage over that of oestradiol-17 beta in suppressing prostatic DNA synthesis at the target tissue level.

Effects of variations in organ culture media and methodology on the proliferative activity of rat ventral prostate and its response to testosterone were investigated quantitatively by using the incorporation of [125I]-iododeoxyuridine (125I-UdR) to monitor DNA synthesis. In serum-free medium, maximal increases in DNA synthesis occurred when testosterone was introduced during the first 48 hours of the culture. Supplementation of the medium with 5% fetal calf serum did not alter the responses in control or testosterone-treated cultures, whereas the addition of insulin (3 micrograms/ml) alone or in combination with serum promoted cell proliferation in testosterone-free cultures and enhanced the stimulatory effect of testosterone. Unlike grid-supported cultures, suspension cultures of rat ventral prostate exhibited increased proliferative activity both in the presence and absence of testosterone. Thus, variations in experimental procedure may account for differences in the proliferative activity of rat prostate between organ culture studies.

Testosterone-induced DNA synthesis in cultured rat ventral prostate was evaluated as an in vitro model for screening the direct effects of anticancer agents on prostatic growth. Optimal conditions for this bioassay, particularly the concentration of testosterone, were established using the inhibitory effects of cyproterone acetate on testosterone-induced 125iododeoxyuridine (I-UdR) uptake as an index of DNA synthesis inhibition. Using 4 X 10(-7) M testosterone, only the highest concentration (X 10(-5) M) of cyproterone acetate inhibited I-UdR uptake and histological observations indicated that this was due to a non-specific cytotoxic effect. In contrast, cyproterone acetate had a dose-dependent inhibitory effect on the proliferative response to 4 X 10(-9) M testosterone. Cyproterone acetate (4 X 10(-7) M) combined with 4 X 10(-9) M testosterone exerted an inhibitory effect on I-UdR uptake and caused epithelial atrophy indicative of androgen deprivation. The results are consistent with similar in vivo studies and confirm the hypothesis that cyproterone acetate acts by directly antagonising testosterone action at the target tissue level. Thus, this in vitro method provides a useful model for screening the direct effects of antiprostatic drugs.

ACTH possesses powerful immuno-suppressive properties and also retards healing by inhibiting fibroblast proliferation and the deposition of collagen. In the present work this hormone was used to test both the 'barrier' and 'immune' hypotheses relating to the failure of CNS regeneration after injury, dosages being administered which had been found to stimulate maximal adrenal production of glucocorticoids in control animals. Although the scarring in the CNS and the systemic humoral and cellular immune responses were significantly depressed, no increase in CNS axon growth was noted, even when large doses of thyroxine were added. We conclude that both 'immune' and 'barrier' hypotheses should be rejected, and that the beneficial effects of thyroxine treatment reported by some workers are not substantiated.

An auto-immune explanation for the failure of axons to regenerate in the central nervous system (CNS) was formulated by Berry & Riches (1974) and Feringa, Johnson & Wendt (1975). Their proposition was that a breakdown in the blood-brain barrier after penetrating injuries to the CNS, or after cerebrovascular accidents, exposed CNS auto-antigens, otherwise sequestered by the blood-brain barrier, to immune surveillance. Specific anti-brain humoral and/or cellular immune responses would thus become mobilised and react with antigens exposed at the wound site. Antibody-antigen complexes so formed could interfere with regeneration by masking molecular pathways along which axons grow, or by inhibiting growth per se by upsetting growth cone mechanisms locally, or by blocking protein synthesis in somata after transportation along the axons. This hypothesis is attractive because it appears to explain the successful regeneration ofmonoaminergic, neurosecretory and laminarlesioned axons, and also provides a basis for a better understanding of the phylogenetic and ontogenetic correlates of regeneration, and the effects of drug treatment on axon growth (Berry & Riches, 1974; Berry, 1979). There are, however, several observations which appear to be inconsistent with this immune hypothesis. Firstly, there has never been a convincing demonstration that embryonic and neonatal axons regenerate (Berry, 1979; Berry & Henry, 1977; Berry et al. 1979a) despite the fact that, in fetal mammals, not only is a competent immune system absent, but many CNS antigens also develop postnatally, possibly after the maturation of the blood-brain barrier (Berry & Riches, 1974). Secondly, peripheral nerves are auto-antigenic, but they readily regenerate, even in the presence of an active experimental peripheral neuritis (Mervart & Kiernan, 1978), and so it seems reasonable to suggest that the failure of CNS regeneration in mammals might equally well be quite unrelated to the auto-antigenicity of CNS tissue. Thirdly, Heinicke & Kieman (1978) have shown that intracerebral skin grafts become liberally innervated by central axons, a result which appears inconsistent with the immune hypothesis since the blood-brain barrier is absent in such grafts (Kiernan, 1978). Lastly, although apparent regeneration has been reported in inmunosuppressed animals using electrophysiological testing (Feringa et al. 1973, 1974, 1975), a subsequent histological survey revealed no unmistakably regenerating axons (Feringa, Kinning, Britten & Vahlsing, 1976). The present paper reports the findings of an extensive study on the effects of a

ABSTRACT Vincristine sulphate has been found to be a more useful metaphase arrest agent than Colcemid. Metaphase accumulation, in isografts of a CBA mammary adenocarcinoma, was linear with time for at least 10 hr after vincristine administration and also independent of drug dose in the range 1–4 mg/kg body weight. A method for evaluating the cell production rate, in histological sections, of a cell population which forms a heterogeneous component of the tissue has been described. The percentage of each tissue component was determined using the Chalkley point count method and the mean number of metaphases in an arbitrary defined field were evaluated. Corrections for percentage composition and sectioning were applied and the cell production rate defined. This was found to be 15.8 ± 0.6 cells per 1000 cells per hour for this tumour.