Energetic heavy ions accelerate differentiation in the descendants of irradiated normal human diploid fibroblasts

Ionizing radiation-induced genomic instability has been demonstrated in a variety of endpoints such as delayed reproductive death, chromosome instability and mutations, which occurs in the progeny of survivors many generations after the initial insult. Dependence of these effects on the linear energy transfer (LET) of the radiation is incompletely characterized; however, our previous work has shown that delayed reductions in clonogenicity can be most pronounced at LET of 108 keV/microm. To gain insight into potential cellular mechanisms involved in LET-dependent delayed loss of clonogenicity, we investigated morphological changes in colonies arising from normal human diploid fibroblasts exposed to gamma-rays or energetic carbon ions (108 keV/microm). Exposure of confluent cultures to carbon ions was 4-fold more effective at inactivating cellular clonogenic potential and produced more abortive colonies containing reduced number of cells per colony than gamma-rays. Second, colonies were assessed for clonal morphotypic heterogeneity. The yield of differentiated cells was elevated in a dose- and LET-dependent fashion in clonogenic colonies, whereas differentiated cells predominated to a comparable extent irrespective of radiation type or dose in abortive colonies. The incidence of giant or multinucleated cells was also increased but much less frequent than that of differentiated cells. Collectively, our results indicate that carbon ions facilitate differentiation more effectively than gamma-rays as a major response in the progeny of irradiated fibroblasts. Accelerated differentiation may account, at least in part, for dose- and LET-dependent delayed loss of clonogenicity in normal human diploid cells, and could be a defensive mechanism that minimizes further expansion of aberrant cells.     

Culture of blastomeres from in vitro-matured,fertilized, and cultured bovine embryos

A culture system was devised to study the differentiation of bovine blastomeres. Blastomeres (2–13 per well) from embryos produced by in vitro maturation, fertilization, and culture of oocytes obtained from slaughterhouse ovaries were cultured for 10 days in 24-well culture plates on feeder layers in blastomere culture medium (BCM: equal parts tissue culture medium 199 and low-glucose Dulbecco's modified Eagle's medium with 10% fetal bovine serum). Ovine embryonic fibroblasts and STO cells were superior to bovine and mouse embryonic fibroblasts as mitotically inactivated feeder cells. Over five studies in which four blastomeres from an embryo were added to each culture well, an average of one colony per well formed from the blastomeres. The colonies continued to grow throughout the culture period, and most colonies resembled trophectoderm in their cellular characteristics, although some cultures contained a mixture of trophectoderm and endoderm. When the number of blastomeres cultured in each well was varied from 2–8, the number of colonies formed was proportional to the number of blastomeres added. Blastomeres from day 5 and day 6 embryos produced fewer colonies than did those from day 4 embryos, perhaps as a result of differentiation and tighter blastomere adhesion resulting in damage during their separation. The absence of serum did not alter the number of colonies formed. A number of growth factors, including LIF, OM, PDGFα, and FGF4, had no effect on the number of colonies, the size of colonies, or their alkaline phosphatase staining score beyond that provided by the feeder layer or serum when present. Blastomeres did not form colonies in the absence of feeder layers. Mol. Reprod. Dev. 48:238–245, 1997. © 1997 Wiley-Liss, Inc.     

The effect of mitotic inhibitors on DNA strand size and radiation-associated break repair in Down syndrome fibroblasts

The effect of mitotic inhibitors on formation and repair of DNA breaks was studied in cultured fibroblasts from patients with Down syndrome in order to investigate the hypothesis that the karyotyping procedure itself may play a role in the increased chromosome breakage seen in these cells after gamma radiation exposure. Using the nondenaturing elution and alkaline elution techniques to examine fibroblasts from Down syndrome patients and from controls, no specific abnormalities in Down syndrome cells could be detected after exposure to mitotic inhibitors, including rate and extent of elution of DNA from filters as well as repair of radiation-induced DNA breaks. In both normal and Down syndrome cell strains, however, exposure to mitotic inhibitors was associated with a decrease in cellular DNA strand size, suggesting the presence of drug-induced DNA strand breaks. The mechanism of increased chromosome sensitivity of Down syndrome cells to gamma radiation remains unknown.     

Studies on the radiosensitivity of cells from patients with basal cell naevus syndrome.

No difference in survival was observed between cultured cells from basal cell naevus syndrome (BCNS) patients and normal controls following exposure of fibroblasts to ionizing radiation. Potential lethal damage repair in BCNS cells, measured by holding experiments, was also no different from normal. G0-irradiated lymphocytes from BCNS patients were found to have a significantly higher level of X-ray-induced chromosome aberrations compared with normals. This increase is, however, small, and, taken together with the survival data, suggests that increased cell killing as a measure of the unusual clinical radiosensitivity is not the major effect of the BCNS gene.     

Abnormal balance between proliferation and apoptotic cell death in fibroblasts derived from keloid lesions

A new culture model was developed to study the role of proliferation and apoptosis in the etiology of keloids. Fibroblasts were isolated from the superficial, central, and basal regions of six different keloid lesions by using Dulbecco's Modified Eagle Medium containing 10% fetal calf serum as a culture medium. The growth behavior of each fibroblast fraction was examined in short-term and long-term cultures, and the percentage of apoptotic cells was assessed by in situ end labeling of fragmented DNA. The fibroblasts obtained from the superficial and basal regions of keloid tissue showed population doubling times and saturation densities that were similar to those of age-matched normal fibroblasts. In contrast, the fibroblasts from the center of the keloid lesions showed significantly reduced doubling times (25.9 +/- 6.3 hours versus 43.5 +/- 6.3 hours for normal fibroblasts) and reached higher cell densities. In long-term culture, central keloid fibroblasts formed a stratified three-dimensional structure, contracted the self-produced extracellular matrix, and gave rise to nodular cell aggregates, mimicking the formation of keloid tissue. Apoptotic cells were detected in both normal and keloid-derived fibroblasts, but their numbers were twofold higher in normal cells compared with all keloid fibroblasts. To examine whether apoptosis mediates the therapeutic effect of ionizing radiation on keloids, the cells were exposed to gamma rays at a dose of 8 Gy. Under these conditions, a twofold increase in the population of apoptotic cells was detected. These results indicate that the balance between proliferation and apoptosis is impaired in keloid fibroblasts, which could be responsible for the formation of keloid tumors. The results also suggest that keloids contain at least two different fibroblast fractions that vary in growth behavior and extracellular matrix metabolism.     

Protein metabolism in the tumour-bearing mouse. Rates of protein synthesis in host tissues and in an Ehrlich ascites tumour at different stages in tumour growth.

The activity of acyl-CoA:cholesterol acyltransferase (ACAT; EC 2.3.1.26) was measured in fibroblast homogenates from Niemann-Pick Type C (NPC) and Type D (NPD) patients to determine whether these cells exhibit similar defects in the regulation of cholesterol esterification. ACAT activity in normal cells cultured in the absence of serum lipoproteins responded rapidly (within 6 h) to the addition of serum and reached peak levels at 12-24 h, whereas little stimulation of activity in NPC cells was observed. In contrast, ACAT activity in NPD fibroblasts (cell lines from four different patients) began to increase between 6 and 12 h after serum addition, reaching levels up to 50% of normal values at 24 h. ACAT activity in NPC and NPD cell extracts could not be stimulated by preincubation with normal cell homogenates, nor was complementation between NPC and NPD homogenates observed. Addition of 25-hydroxycholesterol to fibroblasts cultured in delipidated serum increased ACAT activity for all three cell types, although stimulation in NPD cells was less than that observed in NPC cells. ACAT activity of deoxycholate-solubilized homogenates reconstituted into phosphatidylcholine vesicles was independent of the presence of serum lipoproteins during culture and dependent on cholesterol present in the vesicles for all cell types. However, ACAT activities of mutant fibroblasts in vesicles plus cholesterol were significantly (about 40%) lower than control levels. These results suggest that the metabolic lesions in NPC and NPD cells are biochemically distinct and that both may involve factors in addition to the availability of cholesterol substrate for the ACAT enzyme.     

Response of cultured normal human mammary epithelial cells to X rays

The effect of X rays on the reproductive death of cultured normal human mammary epithelial cells was examined. Techniques were developed for isolating and culturing normal human mammary epithelial cells which provide sufficient cells at second passage for radiation studies, and an efficient clonogenic assay suitable for measuring radiation survival curves. It was found that the survival curves for epithelial cells from normal breast tissue were exponential and had D0 values of about 109-148 rad for 225 kVp X rays. No consistent change in cell radiosensitivity with the age of donor was observed, and no sublethal damage repair in these cells could be detected with the split-dose technique.     

Cholesteatoma Fibroblasts Promote Epithelial Cell Proliferation through Overexpression of Epiregulin

To investigate whether keratinocytes proliferate in response to epiregulin produced by subepithelial fibroblasts derived from middle ear cholesteatoma. Tissue samples were obtained from patients undergoing tympanoplasty. The quantitative polymerase chain reaction and immunohistochemistry were performed to examine epiregulin expression and localization in cholesteatoma tissues and retroauricular skin tissues. Fibroblasts were cultured from cholesteatoma tissues and from normal retroauricular skin. These fibroblasts were used as feeder cells for culture with a human keratinocyte cell line (PHK16-0b). To investigate the role of epiregulin in colony formation by PHK16-0b cells, epiregulin mRNA expression was knocked down in fibroblasts by using short interfering RNA and epiregulin protein was blocked with a neutralizing antibody. Epiregulin mRNA expression was significantly elevated in cholesteatoma tissues compared with that in normal retroauricular skin. Staining for epiregulin was more intense in the epithelial cells and subepithelial fibroblasts of cholesteatoma tissues than in retroauricular skin. When PHK16-0b cells were cultured with cholesteatoma fibroblasts, their colony-forming efficiency was 50% higher than when these cells were cultured with normal skin fibroblasts. Also, knockdown of epiregulin mRNA in cholesteatoma fibroblasts led to greater suppression of colony formation than knockdown in skin fibroblasts. Furthermore, the colony-forming efficiency of PHK16-0b cells was significantly reduced after treatment with an epiregulin neutralizing antibody in co-culture with cholesteatoma fibroblasts, but not in co-culture with skin fibroblasts. These results suggest that keratinocyte hyperproliferation in cholesteatoma is promoted through overexpression of epiregulin by subepithelial fibroblasts via epithelial–mesenchymal interactions, which may play a crucial role in the pathogenesis of middle ear cholesteatoma.     

Cardiac Fibroblasts Regulate Sympathetic Nerve Sprouting and Neurocardiac Synapse Stability

Sympathetic nervous system (SNS) plays a key role in cardiac homeostasis and its deregulations always associate with bad clinical outcomes. To date, little is known about molecular mechanisms regulating cardiac sympathetic innervation. The aim of the study was to determine the role of fibroblasts in heart sympathetic innervation. RT-qPCR and western-blots analysis performed in cardiomyocytes and fibroblasts isolated from healthy adult rat hearts revealed that Pro-Nerve growth factor (NGF) and pro-differentiating mature NGF were the most abundant neurotrophins expressed in cardiac fibroblasts while barely detectable in cardiomyocytes. When cultured with cardiac fibroblasts or fibroblast-conditioned medium, PC12 cells differentiated into/sympathetic-like neurons expressing axonal marker Tau-1 at neurites in contact with cardiomyocytes. This was prevented by anti-NGF blocking antibodies suggesting a paracrine action of NGF secreted by fibroblasts. When co-cultured with cardiomyocytes to mimic neurocardiac synapse, differentiated PC12 cells exhibited enhanced norepinephrine secretion as quantified by HPLC compared to PC12 cultured alone while co-culture with fibroblasts had no effect. However, when supplemented to PC12-cardiomyocytes co-culture, fibroblasts allowed long-term survival of the neurocardiac synapse. Activated fibroblasts (myofibroblasts) isolated from myocardial infarction rat hearts exhibited significantly higher mature NGF expression than normal fibroblasts and also promoted PC12 cells differentiation. Within the ischemic area lacking cardiomyocytes and neurocardiac synapses, tyrosine hydroxylase immunoreactivity was increased and associated with local anarchical and immature sympathetic hyperinnervation but tissue norepinephrine content was similar to that of normal cardiac tissue, suggesting depressed sympathetic function. Collectively, these findings demonstrate for the first time that fibroblasts are essential for the setting of cardiac sympathetic innervation and neurocardiac synapse stability. They also suggest that neurocardiac synapse functionality relies on a triptych with tight interaction between sympathetic nerve endings, cardiomyocytes and fibroblasts. Deregulations of this triptych may be involved in pathophysiology of cardiac diseases.     

Ecological characterization of coral growth anomalies on Porites compressa in Hawai��i

Bulbous skeletal structures with associated aberrant corallites have been abundant on Porites compressa in Kāne‘ohe Bay, O‘ahu, Hawai‘i, for at least the last 19 years. These growth anomalies (GA) appear in the summer in shallow (<3 m) water on some, but not on all colonies. GA-free branches, collected from colonies with GAs, produced GAs when cultured in outdoor flow-thru aquaria. Normal branches, whose tissues were continuous with those of GAs, grew in length much more slowly than normal branches from the same colony that were not connected with a GA, suggesting that there is a translocation of materials from normal tissue to GAs. Small experimental colonies that were either exposed to, or protected from, UV radiation did not differ in their rate of GA formation. GAs had a lower probability of survival than normal branches. This characteristic, in combination with their effect on the growth of normal branches and other reported deficiencies in the tissues of growth anomalies (e.g., reduced or failed reproduction), suggests that GA-bearing colonies of this species have reduced fitness.     

Establishment of keratinocyte colonies from small-sized cervical intraepithelial neoplasia specimens

The size of human cervical intraepithelial neoplasia (CIN) biopsies is usually very small and standard methods do not allow an adequate number of keratinocytes to be isolated for culturing purposes. In this study, a new approach to establish keratinocyte cultures from small CIN a tissue fragments was developed. Neoplastic specimens and corresponding normal tissues, which were used as controls, were digested with collagenase. Tissue‐derived fibroblasts and keratinocytes were co‐cultured in calcium and serum medium. Single keratinocyte colonies from primary cultures were expanded using a culture medium optimized in our laboratory. Primary keratinocyte colonies, as well as expanded colonies, were tested for epithelial and cervical markers such as 5, 14, 17, and 19 keratins, and p63 by immunofluorescence. Our results indicate that a variable number of primary keratinocyte colonies could be detected in neoplastic cultures, depending on the grade of cervical lesions from which the colonies originated. Single colonies, when cultured with our new medium, grew at a high rate with uniform size and morphology for some passages. Epithelial and p63 markers were expressed in keratinocyte colonies, as well as in expanded colonies. In conclusion, our study reports a rapid and easy culturing system which enables keratinocyte colonies from minute cervical tumor tissues to be obtained. Moreover, using the new culture medium, keratinocyte colonies can be expanded at a high proliferative rate. J. Cell. Physiol. 227: 3787–3795, 2012. © 2012 Wiley Periodicals, Inc.     

Fibroblasts from patients with inherited predisposition to retinoblastoma exhibit normal sensitivity to the mutagenic effects of ionizing radiation

Retinoblastoma (RB) is a cancer of the retina which characteristically occurs in early childhood. Bilateral RB is an inherited form of this disease. Such patients are at greatly increased risk of subsequently developing second tumors in mesenchymal tissue, especially in areas exposed to ionizing radiation therapy. Fibroblasts from bilateral RB patients have been reported to be more sensitive than normal fibroblasts to the cytotoxic effects of ionizing radiation. Because xeroderma pigmentosum patients have a hereditary predisposition to UV-induced cancer and the cells of such patients are abnormally sensitive to the cytotoxic and mutagenic effects of UV radiation, we compared fibroblasts from 6 bilateral RB patients and 3 normal individuals for their sensitivity to the mutagenic effects of cobalt 60, using resistance to 6-thioguanine (TG) as the genetic marker. The results showed no statistically significant difference between the two types of cell lines. The slope of the weighted least squares line representing the frequency of TG-resistant cells induced in the RB populations as a function of dose was 17 +/- 6 (S.E.)/10(6) cells/Gy with an intercept of 0.09 Gy; that for the normal cells was 17 +/- 7/10(6) cells/Gy with an intercept of 0.14 Gy. We also compared 8 bilateral RB cell lines and 9 age-matched normal cell lines for their sensitivity to the cytotoxic effect of 60Co, using survival of colony-forming ability. The cloning efficiency of the unirradiated RB cell lines ranged from 22% to 76% with an average of 52%; that of the normal cell lines from 21% to 89% with an average of 64%. The results showed the RB cells were somewhat more sensitive than the normal cells. The mean D0 for the RB cell lines ranged from 0.99 +/- 0.01 (S.E.) to 1.69 +/- 0.04 Gy with a weighted average of 1.44 +/- 0.08 Gy; that of the normal cell lines ranged from 1.42 +/- 0.17 to 2.24 +/- 0.10 Gy, with a weighted average of 1.79 +/- 0.11 Gy. The difference in means was estimated to be 0.34 +/- 0.14. The mean for the RB cell lines is statistically significantly lower than the mean for the normal cell lines, at a significance level ca. 1%.     

Fibroblasts from Retinoblastoma patients: Enhanced growth in fetal calf serum and a normal response to ionizing radiation

Retinoblastoma is a rare malignant eye tumor found almost exclusively in young children. In 30% of cases, the tumor is bilateral and is inherited as an autosomal dominant trait. In such patients, all of the cells in the body must carry the mutation predisposing to retinoblastoma. To search for the expression of the gene in cells outside the retina, we have studied several in vitro properties of skin fibroblasts from patients with bilateral retinoblastoma. Measurement in low concentrations of fetal calf serum of the initial growth rate and the plating efficiency show that fibroblasts from retinoblastoma donors grow significantly better than those from normal donors. However, we were unable to confirm the results of other investigators that fibroblasts from donors with bilateral retinoblastoma are unusually sensitive to ionizing radiation. In family studies, skin fibroblasts from normal siblings had the same radiation sensitivity as fibroblasts from sibling with retinoblastoma.     

Induction of MiR-21 by Stereotactic Body Radiotherapy Contributes to the Pulmonary Fibrotic Response

Radiation-induced lung fibrosis, the most serious effect of lung cancer radiotherapy on normal tissue, remains a major technical obstacle to the broader application of radiotherapy to patients with lung cancer. This study describes the use of an image-guided irradiation system in mice mimicking stereotactic body radiotherapy (SBRT) to examine the molecular features of chronic fibrotic response after radiation injury. MicroRNA (miR) array analysis of injured pulmonary tissue identified a set of miRs whose expression was significantly increased in damaged lung tissue. In particular, miR-21 expression was increased at the radiation injury site, concurrent with collagen deposition. Although the inhibition of miR-21 by its specific inhibitor anti-miR-21 only marginally affected endothelial-mesenchymal transition (EndMT) in lung endothelial cells, this inhibition significantly reduced collagen synthesis in lung fibroblasts. Furthermore, ectopic expression of miR-21 was sufficient to promote a fibrotic response in lung fibroblasts, enhancing Smad2 phosphorylation concurrent with Smad7 downregulation. These findings indicate that the induction of miR-21 expression is responsible for fibrotic responses observed in mesenchymal cells at the injury site through the potentiation of TGF-β signaling. Local targeting of miR-21 at the injured area could have potential therapeutic utility in mitigating radiation-induced lung fibrosis.     

Abnormal myo-inositol and phospholipid metabolism in cultured fibroblasts from patients with ataxia telangiectasia

Ataxia telangiectasia (AT) is a complex autosomal recessive disorder that has been associated with a wide range of physiological defects including an increased sensitivity to ionizing radiation and abnormal checkpoints in the cell cycle. The mutated gene product, ATM, has a domain possessing homology to phosphatidylinositol-3-kinase and has been shown to possess protein kinase activity. In this study, we have investigated how AT affects myo-inositol metabolism and phospholipid synthesis using cultured human fibroblasts. In six fibroblast lines from patients with AT, myo-inositol accumulation over a 3-h period was decreased compared to normal fibroblasts. The uptake and incorporation of myo-inositol into phosphoinositides over a 24-h period, as well as the free myo-inositol content was also lower in some but not all of the AT fibroblast lines. A consistent finding was that the proportion of 32P in total labeled phospholipid that was incorporated into phosphatidylglycerol was greater in AT than normal fibroblasts, whereas the fraction of radioactivity in phosphatidic acid was decreased. Turnover studies revealed that AT cells exhibit a less active phospholipid metabolism as compared to normal cells. In summary, these studies demonstrate that two manifestations of the AT defect are alterations in myo-inositol metabolism and phospholipid synthesis. These abnormalities could have an effect on cellular signaling pathways and membrane production, as well as on the sensitivity of the cells to ionizing radiation and proliferative responses.     

Inhibition of psoriatic cell proliferation in in vitro skin models by amiprilose hydrochloride

Summary Amiprilose hydrochloride, a 3-substituted glucose derivative, was found to inhibit the proliferation of human fibroblasts and keratinocytes originating from psoriatic lesions. Fibroblasts and keratinocytes were obtained from skin biopsies of normal donors, and from the biopsies of active/involved and uninvolved sites of psoriatic donors. The cells were cultured as monolayers or as components of tissue equivalent models. Keratinocytes and fibroblasts originating from biopsies of psoriatically involved areas were shown to proliferate at a significantly higher rate than those derived from uninvolved areas. The antiproliferative effect of amiprilose hydrochloride was not observed with normal keratinocytes or fibroblasts from the skin of healthy donors or from uninvolved areas of psoriatic donors. Amiprilose hydrochloride was not cytotoxic to any of these cells at levels below 0.1%. The combination of the low cytotoxicity and the selective antiproliferative effect indicates that this compound may be a useful antipsoriatic agent. The use of monolayer cultures and tissue equivalent models in this study illustrates the utility of such a progressive strategy in the evaluation of potential topical pharmaceuticals. Supported in part by research grants from the National Institutes of Health (AG01274), The R.A. Welch Foundation (B-0502), The Texas, Advanced Technology and Research Program (Wound Healing and Aging #2147), and Greenwich Pharmaceuticals Inc. R. W. G. is the recipient of a MERIT Award from the National Institute on Aging.     

Defects in the processing of procollagen to collagen are demonstrable in cultured fibroblasts from patients with the Ehlers-Danlos and osteogenesis imperfecta syndromes

This is a study of the processing of procollagen to collagen in cultures of skin and tendon fibroblasts. Processing was markedly increased by growing cells for 2-4 days postconfluence and then adding ascorbate to the medium for 2 days prior to labeling with [3H] proline. With this system, more than two-thirds of the pro-alpha chains of type I procollagen in the culture medium, and more than 90% of those in the cell layer, were rapidly processed to pC-alpha, pN-alpha, or alpha chains. Purified, exogenous procollagen was also rapidly processed in cell-free culture medium. The results showed for the first time that exogenous procollagen can be processed in conditioned cell-free medium. The system was then used to compare the processing of procollagen in the medium of normal fibroblasts, cells from one bovine and four human variants of osteogenesis imperfecta, and those from eight human variants of the Ehlers-Danlos syndrome. The cells could be divided into three groups, based on their ability to process type I procollagen: normal, consistently slow, and very slow. The cause of the decreased processing was shown to be associated with either a mutation causing a shortening of an alpha chain or decreased activity of procollagen N-proteinase in cell-free culture medium. Decreased processing of procollagen to collagen occurred with cultured fibroblasts from patients with different forms of both osteogenesis imperfecta and Ehlers-Danlos syndrome. Both of these disease syndromes are associated with abnormalities in the structure or metabolism of procollagen in fibrous connective tissues, bones, and teeth. The results show that defects in the structure, synthesis, or processing of procollagen are readily demonstrated with cultured fibroblasts.     

Factors derived from mouse embryonic stem cells promote self-renewal of goat embryonic stem-like cells

Goat embryonic stem (ES)-like cells could be isolated from primary materials-inner cell masses (ICMs) and remain undifferentiated for eight passages in a new culture system containing mouse ES cell conditioned medium (ESCCM) and on a feeder layer of mouse embryo fibroblasts (MEFs). However, when cultured in medium without mouse ESCCM, goat ES-like cells could not survive for more than three passages. In addition, no ES-like cells could be obtained when ICMs were cultured on goat embryo fibroblasts or the primary materials-whole goat blastocysts were cultured on MEFs. Goat ES-like cells isolated from ICMs had a normal karyotype and highly expressed alkaline phosphatase. Multiple differentiation potency of the ES-like cells was confirmed by differentiation into neural cells and fibroblast-like cells in vitro. These results suggest that mouse ES cells might secrete factors playing important roles in promoting goat ES-like cells' self-renewal, moreover, the feeder layers and primary materials could also influence the successful isolation of goat ES-like cells.     

Differential sensitivities to gamma radiation of human bladder and testicular tumour cell lines

Gamma radiation sensitivities of continuous cell lines from nine human tumours were measured, comparing four derived from transitional cell carcinomas of the bladder with five from non-seminomatous germ cell tumours of the testis. The testicular cells were significantly more radiosensitive than the bladder cells, corresponding to the response to therapy of these tumour types in patients. These observations indicate that radiosensitivity is retained in vitro and is an inherent property of the testicular tumour cells. These gamma radiation sensitivities were compared with those of SV40-transformed fibroblasts derived from a normal individual and one with the heritable disease, ataxia-telangiectasia (A-T). The bladder cells had gamma radiation sensitivities similar to that of the SV40-transformed normal line. The testicular cells were hypersensitive to gamma radiation, although not as sensitive as the SV40-transformed A-T line. A-T cells, unlike those derived from normal individuals, continue to synthesize DNA at a normal rate following radiation exposure, prompting a comparison of the kinetics of DNA synthesis in three bladder and three testicular tumour cell lines. One of the bladder and two testicular lines showed a reduced inhibition when compared to the other tumour cell lines and the SV40-transformed normal line. Thus there was no clear association between DNA synthesis inhibition and radiosensitivity.     

Zinc protects against ultraviolet A1-induced DNA damage and apoptosis in cultured human fibroblasts

Ultraviolet Al (UVA1) radiation generates reactive oxygen species and the oxidative stress is known as a mediator of DNA damage and of apoptosis. We exposed cultured human cutaneous fibroblasts to UVA1 radiation (wavelengths in the 340–450-nm range with emission peak at 365 nm) and, using the alkaline unwinding method, we showed an immediate significant increase of DNA strand breaks in exposed cells. Apoptosis was determined by detecting cytoplasmic nucleosomes (enzyme-linked immunosorbent assay method) at different time points in fibroblasts exposed to different irradiation doses. In our conditions, UVA1 radiation induced an early (8 h) and a delayed (18 h) apoptosis. Delayed apoptosis increased in a UVA dosedependent manner. Zinc is an important metal for DNA protection and has been shown to have inhibitory effects on apoptosis. The addition of zinc (6.5 mg/L) as zinc chloride to the culture medium significantly decreased immediate DNA strand breaks in human skin fibroblasts. Moreover, zinc chloride significantly decreased UVA1-induced early and delayed apoptosis. Thus, these data show for the first time in normal cutaneous cultured cells that UVA1 radiation induces apoptosis. This apoptosis is biphasic and appears higher 18 h after the stress. Zinc supplementation can prevent both immediate DNA strand breakage and early and delayed apoptosis, suggesting that this metal could be of interest for skin cell protection against UVA1 irradiation.