Identification of KIN (KIN17), a human gene encoding a nuclear DNA-binding protein, as a novel component of the TP53-independent response to ionizing radiation.

Ionizing radiation elicits a genetic response in human cells that allows cell survival. The human KIN (also known as KIN17) gene encodes a 45-kDa nuclear DNA-binding protein that participates in the response to UVC radiation and is immunologically related to the bacterial RecA protein. We report for the first time that ionizing radiation and bleomycin, a radiomimetic drug, which produce single- and double-strand breaks, increased expression of KIN in human cells established from tumors, including MeWo melanoma, MCF7 breast adenocarcinoma, and ATM+ GM3657 lymphoblast cells. KIN expression increased rapidly in a dose-dependent manner after irradiation. Under the same conditions, several genes controlled by TP53 were induced with kinetics similar to that of KIN. Using the CDKN1A gene as a marker of TP53 responsiveness, we analyzed the up-regulation of KIN and showed that is independent of the status of TP53 and ATM. In contrast, the presence of a dominant mutant for activating transcription factor 2 (ATF2) completely abolished the up-regulation of KIN. Our results suggest a role for ATF2 in the TP53-independent increase in KIN expression after gamma irradiation.

Molecular cloning and characterization of the human KIN17 cDNA encoding a component of the UVC response that is conserved among metazoans.

We describe the cloning and characterization of the human KIN17 cDNA encoding a 45 kDa zinc finger nuclear protein. Previous reports indicated that mouse kin17 protein may play a role in illegitimate recombination and in gene regulation. Furthermore, overproduction of mouse kin17 protein inhibits the growth of mammalian cells, particularly the proliferation of human tumour-derived cells. We show here that the KIN17 gene is remarkably conserved during evolution. Indeed, the human and mouse kin17 proteins are 92.4% identical. Furthermore, DNA sequences from fruit fly and filaria code for proteins that are 60% identical to the mammalian kin17 proteins, indicating conservation of the KIN17 gene among metazoans. The human KIN17 gene, named (HSA)KIN17, is located on human chromosome 10 at p15-p14. The (HSA)KIN17 RNA is ubiquitously expressed in all the tissues and organs examined, although muscle, heart and testis display the highest levels. UVC irradiation of quiescent human primary fibroblasts increases (HSA)KIN17 RNA with kinetics similar to those observed in mouse cells, suggesting that up-regulation of the (HSA)KIN17 gene after UVC irradiation is a conserved response in mammalian cells. (HSA)kin17 protein is concentrated in intranuclear focal structures in proliferating cells as judged by indirect immunofluorescence. UVC irradiation disassembles (HSA)kin17 foci in cycling cells, indicating a link between the intranuclear distribution of (HSA)kin17 protein and the DNA damage response.

Short term pretreatment with medroxyprogesterone and testosterone may potentiate irradiation damage to spermatogenesis in rats.

BACKGROUND: Hormonal treatments lasting 2-6 months inhibit spermatogenesis in men and have been proposed as germ cell protection against anticancer therapy. Because it is unthinkable to delay anticancer treatments, the authors investigated the protection afforded against irradiation of rats by 22 days of hormonal pretreatment. METHODS: Adult Sprague-Dawley rats were assigned to an untreated control group (C) or to one of 5 treatments: medroxyprogesterone acetate plus testosterone only (M), 3 or 5 gray of irradiation (R3 and R5), or hormonal treatment prior to 3 or 5 gray of irradiation (MR3 and MR5). Mating trials were conducted 1, 24, 45, 65, 86, and 109 days after treatment. At 122 days, genital organ weights, testis histology, and epididymal spermatozoa were evaluated. RESULTS: Irradiation reduced sperm production and had a clastogenic effect on postmeiotic germ cells. No protective effect of steroid treatment was observed. Moreover, testis weight, tubule diameter, the repopulating index, and the sperm head count decreased more in the MR5 group than in the R5 group. Mating tests showed decreases in positive vaginal smears and fertility at both 45 and 65 days, and an increase in resorption at 109 days. CONCLUSIONS: These results indicate that hormonal pretreatment potentiates irradiation damage to germ cells, especially stem cells, as regards survival and genomic alterations, probably because of increased lipoperoxidation of late spermatids.

The nuclear concentration of kin17, a mouse protein that binds to curved DNA, increases during cell proliferation and after UV irradiation.

UV-irradiation induces, in mammalian cells, the expression of a set of genes known as the 'UV-response', which may be reminiscent of the bacterial response, called SOS system. The multifunctional protein RecA controls the expression of the SOS genes. We report the expression profile of a mouse gene conserved among mammals, called Kin17, that codes a DNA-binding protein of undetermined biochemical activity and which shares epitopes with the bacterial RecA protein. We demonstrate that the level of Kin17 RNA was 5-fold higher in mid-S phase of serum-stimulated BALB/c 3T3 fibroblasts than in quiescent cells. Cells in S-phase displayed a high level of kin17 protein with a marked nuclear localisation. The maximal level of Kin17 RNA was observed 18 h after serum stimulation, indicating that Kin17 gene is a new member of the late growth-related genes. The accumulation of kin17 protein during cell proliferation follows the increase in Kin17 RNA and correlates with DNA synthesis, which suggests a possible role of kin17 protein in a transaction related to DNA-replication. In quiescent fibroblasts, a 3-fold increase in Kin17 RNA was seen 13 h after UV irradiation. In parallel, kin17 protein accumulated in the nucleus, which suggests that it might be required after the stress produced by UV irradiation.

Lead affects steroidogenesis in rat Leydig cells in vivo and in vitro.

Lead is known to impede the male reproductive function, however, the mechanisms through which the adverse effects are mediated are not clearly elucidated. In order to get insight into those mechanisms, we have examined the effects of lead on the biosynthesis of steroid hormones by Leydig cells in the rat. To determine whether lead has a direct action on Leydig cells, we have compared the concentrations of testosterone secreted by Leydig cells in ex vivo experiments after animals had been injected with high doses of lead and in vitro experiments with Leydig cells from normal rats maintained in culture in presence or absence of lead. In ex vivo experiments male Spargue-Dawley rats were injected i.p. with lead acetate (8 mg lead/kg/day, 5 days a week for 5 weeks) or with sodium acetate. Testosterone production by Leydig cells isolated and maintained in culture for 48 h was then assessed under basal conditions or after stimulation by human chorionic gonadotrophin (hCG). Both basal and hCG-stimulated testosterone production dropped by 59% and 37%, respectively, with Leydig cells from lead-exposed rats. For in vitro experiments, cultures of Leydig cells from control rats were exposed to various concentrations of lead acetate for different periods. Dose and time-dependent reductions of testosterone level were observed in the culture medium. The effective doses of hCG for maximal and half-maximal testosterone production did not change, indicating that the sensitivity of Leydig cells to hCG was not impaired by exposure to lead in vitro. Progesterone production was also decreased after this exposure. The negative effect of lead on testosterone and progesterone production was correlated with the lower expression of the enzymes cytochromes P450scc (CYP11A1) and P450c17 (CYP17) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) involved in steroid hormone biosynthesis, as shown by immunohistochemistry. Ultrastructural alterations of the smooth endoplasmic reticulum observed after lead administration might be correlated with the lower expression of the microsomal enzymes P450c17 and 3 beta-HSD. Our results indicate that lead can adversely affect the Leydig cell function by impairing directly steroidogenesis.

Impairment of testicular endocrine function after lead intoxication in the adult rat.

To clarify the mechanism of the action of lead on male reproductive function, adult male rats were injected intraperitoneally (i.p.) with lead acetate (8 mg/kg/day of lead), 5 days a week for 35 days. Despite this high dose, germ cells and Sertoli cells did not appear to be major targets of lead. However, lead determination in the reproductive organs showed that the accessory sex glands are such a target. Epididymal function was unchanged. In lead-exposed rats, plasma and testicular testosterone dropped by about 80%, but plasma luteinizing hormone (LH) only dropped by 32%. After luteinizing hormone releasing hormone (LHRH) stimulation of the pituitary, the plasma LH level reached the control one, but plasma testosterone remained significantly reduced by 37%. The sharp decrease in the testosterone:LH ratio in lead-exposed rats, combined with the significant reduction of intertubular tissue volume in the testes, indicate impaired Leydig cell function.