Biomedical subjects

M Stefanini

Publications and source records attributed to M Stefanini.

At least 55 records · Page 3Linked to original sources

PubMed1997

A variant of the Nijmegen breakage syndrome with unusual cytogenetic features and intermediate cellular radiosensitivity.

We report the first Italian case of Nijmegen breakage syndrome (NBS). The proband is an immunodeficient, microcephalic, 11 year old boy with a "bird-like" face. He developed a T cell rich B cell lymphoma. Spontaneous chromosomal instability was detected in T and B lymphocytes and fibroblasts; chromosomes 7 and 14 were only sporadically involved in the rearrangements and no clonal abnormality was present. The patient appeared to be sensitive both to ionising radiation and to bleomycin, although his sensitivity did not reach the level of AT reference cells. After bleomycin treatment, inhibition of DNA synthesis was low when compared with normal cells, but higher than observed in an AT reference strain. Moreover, cell cycle analysis, after drug exposure, showed a progressive reduction in the percentage of S phase cells, but the G1 arrest, found in normal cells, was not observed. On clinical evaluation our patient shares features with NBS subjects, but cytogenetic and cell biological data do not completely overlap with those reported in Nijmegen breakage syndrome. The ethnic origin of our patient might account for these differences, as expression of different allelic forms at the NBS locus.

Antigens, Surface↗

PubMed1996

A CHO mutant, UV40, that is sensitive to diverse mutagens and represents a new complementation group of mitomycin C sensitivity.

A new mitomycin C (MMC)-sensitive rodent line, UV40, has been identified in the collection of ultraviolet light- (UV-) sensitive mutants of Chinese hamster ovary (CHO) cells isolated at the previous Facility for Automated Experiments in Cell Biology (FAECB). It was isolated from an UV mutant hunt using mutagenesis of AA8 cells with the DNA intercalating frameshift mutagen ICR170. It is complemented by CHO-UV-1, irsl, irs3, irslSF, MC5, V-C8 and V-H4 with respect to its MMC sensitivity based on cell survival. Despite having approx. 4 X normal UV sensitivity and increased sensitivity to UV inhibition of DNA replication, it has near-normal incision kinetics of UV irradiated DNA, and normal (6-4) photoproducts removal. It also is not hypermutable by UV, and shows near normal levels of UV inhibition of RNA synthesis. UV40 also has approx. 11 x .10 x .5 x and 2 x AA8 sensitivity to MMC, ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), and X-rays, respectively. Thus, its defect apparently does not involve nucleotide excision repair but rather another process, possibly in replicating past lesions. The spontaneous chromosomal aberration frequency is elevated to 20% in UV40, and the baseline frequency of sister chromatid exchange is also approximately 4-fold increased. The phenotype of UV40 appears to differ from all other rodent mutants that have so far been described.

Adenine↗

PubMed1996

Sensitivity of CHO mutant cell lines with specific defects in nucleotide excision repair to different anti-cancer agents.

Nucleotide excision repair (NER) is one of the major DNA repair systems in mammalian cells, able to remove a broad spectrum of unrelated lesions. In this report the role of ERCC (excision repair cross-complementing) 1, ERCC2, ERCC3, ERCC4, and ERCC6 genes in removing the lesions caused by alkylating agents with different structures and mechanisms of action has been studied using UV-sensitive DNA repair-deficient mutant CHO cell lines. We confirmed that ERCC1 and ERCC4 play a role in the repair of cis-diamminedichloroplatinum (DDP)- and Melphalan (L-PAM)-induced DNA damage, while a marginal role of ERCC2 and ERCC3 in the cellular response to DDP and L-PAM treatment has been observed. Treatment with methylating agents (DM and MNNG) showed a lack of a preferential cytotoxicity between the parental and the different NER. deficient cell lines, emphasizing the importance of other repair systems such as 3-methyladenine glycosylase and O6 alkyl-guanine-DNA-alkyl-transferase. ERCC1, ERCC2, ERCC3 and ERCC4, but not ERCC6 gene products seem to be involved in removing the lesions caused by Tallimustine and CC1065, minor groove alkylating agents that alkylate N3 adenine in a sequence-specific manner. ERCC6-deficient cells were as sensitive as the parental cell line to all the cytotoxic drugs tested, except DDP. These data emphasize the importance of the CHO mutant cell lines with specific defects in the DNA repair system for investigating the mechanism of action of different anti-cancer agents.

Animals↗

PubMed1996

Genetic analysis of twenty-two patients with Cockayne syndrome.

Cockayne syndrome (CS) is an autosomal recessive disorder with dwarfism, mental retardation, sun sensitivity and a variety of other features. Cultured CS cells are hypersensitive to ultraviolet (UV) light, and following UV irradiation, CS cells are unable to restore RNA synthesis rates to normal levels. This has been attributed to a specific deficiency in CS cells in the ability to repair damage in actively transcribed regions of DNA at the rapid rate seen in normal cells. We have used the failure of recovery of RNA synthesis, following UV irradiation of CS cells, in a complementation test. Cells of different CS donors are fused. Restoration of normal RNA synthesis rates in UV-irradiated heterodikaryons indicates that the donors are in different complementation groups, whereas a failure to effect this recovery implies that they are in the same group. In an analysis of cell strains from 22 CS donors from several countries and different racial groups, we have assigned five cell strains to the CS-A group and the remaining 17 to CS-B. No obvious racial, clinical or cellular distinctions could be made between individuals in the two groups. Our analysis will assist the identification of mutations in the recently cloned CSA and CSB genes and the study of structure-function relationships.

Adult↗

PubMed1996

Isolation of the synchronized A spermatogonia from adult vitamin A-deficient rat testes.

A method for isolating A spermatogonia from the adult vitamin A-deficient (VAD) rat testis is described. After removal, the testes were decapsulated and tubules were dissected. An enzymatic digestion with collagenase, hyaluronidase, and trypsin was performed first to eliminate most of the interstitial cells. A second digestion with collagenase and hyaluronidase was performed to obtain a cell suspension with a high number of A spermatogonia. The cell suspension was further enriched with A spermatogonia by preplating on peanut agglutinin and separating on a discontinuous Percoll gradient. By this procedure, purification of the suspension to 70-90% A spermatogonia was obtained. In the seminiferous tubules of the VAD rats, only Sertoli cells, A spermatogonia, and some preleptotene spermatocytes are present. In our rats, the A spermatogonia are almost all arrested in the G1 phase of the cell cycle before the S phase of A1 spermatogonia, and presumably before their differentiation into A1 spermatogonia. After administration of vitamin A, spermatogenesis starts synchronously from these A spermatogonia. The isolation of these synchronized A spermatogonia opens ways to investigate the regulation of differentiation and proliferation of A spermatogonia and the biochemical characteristics of the subsequent types of A spermatogonia.

Animals↗

PubMed1996

DNA repair and ultraviolet mutagenesis in cells from a new patient with xeroderma pigmentosum group G and cockayne syndrome resemble xeroderma pigmentosum cells.

Xeroderma pigmentosum (XP)/Cockayne syndrome (CS) complex is a combination of clinical features of two rare genetic disorders in one individual. A sun-sensitive boy (XP20BE) who had severe symptoms of CS, with dwarfism, microcephaly, retinal degeneration, and mental impairment, had XP-type pigmentation and died at 6 y with marked cachexia (weight 14.5 lb) without skin cancers. We evaluated his cultured cells for characteristic CS or XP DNA-repair abnormalities. The level of ultraviolet (UV)-induced unscheduled DNA synthesis was less than 5% of normal, characteristic of the excision-repair defect of XP. Cell fusion studies indicated that his cells were in XP complementation group G. His cells were hypersensitive to killing by UV, and their post-UV recovery of RNA synthesis was abnormally low, features of both CS and XP. Post-UV survival of plasmid pSP189 in his cells was markedly reduced, and post-UV plasmid mutation frequency was higher than with normal cells, as in both CS and XP. Sequence analysis of the mutated plasmid marker gene showed normal frequency of plasmids with multiple base substitutions, as in CS, and an abnormally increased frequency of G:C-->A:T mutations, a feature of XP. Transfection of UV-treated pRSVcat with or without photoreactivation revealed that his cells, like XP cells, could not repair either cyclobutane pyrimidine dimers or non-dimer photoproducts. These results indicate that the DNA-repair features of the XP20BE (XP-G/CS) cells are phenotypically more like XP cells than CS cells, whereas clinically the CS phenotype is more prominent than XP.

Cell Survival↗

PubMed1995

Defects in the DNA repair and transcription gene ERCC2 in the cancer-prone disorder xeroderma pigmentosum group D.

Xeroderma pigmentosum (XP) is a sun-sensitive, cancer-prone genetic disorder characterized by a defect in nucleotide excision repair. The human nucleotide excision repair and transcription gene ERCC2 is able to restore survival to normal levels after exposure to UV light in XP complementation group D cells. No enhancement of UV survival is seen in groups C, E, F, or G. XP-CS-2 cells are complemented by ERCC2, confirming the reassignment to group D of this combined XP/Cockayne's syndrome patient. Nucleotide sequence analysis of the ERCC2 cDNA from five XP group D cell strains [XP6BE(SV40), XP17PV, XP102LO, A31-27 (a HeLa/XP102LO hybrid), and XP-CS-2] revealed mutations predominantly affecting previously identified functional domains. The mutations include base substitutions resulting in amino acid substitutions, deletions due to splicing alterations, and defects in expression. XP6BE(SV40), XP17PV, XP102LO, and A31-27 all have one allele with an Arg683 to Trp substitution within the putative nuclear location signal. The genetic disorder trichothiodystrophy (which is not cancer-prone) can also result from mutations in the ERCC2 gene, some of which are the same as those found in XP-D. The various clinical presentations can be correlated with the particular mutations found in the ERCC2 locus.

Adult↗

PubMed1995

Different removal of ultraviolet photoproducts in genetically related xeroderma pigmentosum and trichothiodystrophy diseases.

To understand the heterogeneity in genetic predisposition to skin cancer in different nucleotide excision repair-deficient human syndromes, we studied repair of cyclobutane pyrimidine dimers (CPDs) and of pyrimidine(6-4)pyrimidone (6-4PP) photoproducts in cells from trichothiodystrophy (TTD) patients. TTD is not associated with increased incidence of skin cancer, although 50% of the patients are photosensitive and carry a defect in the nucleotide excision repair pathway, similar to Xeroderma pigmentosum patients. However, in striking contrast to TTD, Xeroderma pigmentosum is highly prone to cancer. To address this apparent paradox, two types of studies were conducted: (a) reactivation of UV-irradiated plasmids harboring actively transcribed reporter genes, with or without photolyase treatment before transfection of SV40-transformed fibroblasts; and (b) the kinetics of removal of UV-induced CPDs and 6-4PPs in genomic DNA by immunoblot analysis using lesion-specific mAbs in SV40-transformed and untransformed fibroblasts representative of all genetic TTD complementation groups. Results showed that all cell lines from photosensitive TTD patients efficiently express Cat or luciferase genes in transfected plasmids carrying non-CPD lesions, including 6-4PP, and display wild-type or near-wild-type (50-70% in 3 cell lines) 6-4PP repair in the overall genome after immunoblot analysis. However, CPD lesions (the repair of which is defective in the overall genome) also block the expression of the reporter gene in transfected plasmids. Two cell lines from nonphotosensitive TTD patients showed wild-type levels of repair for both photoproducts in overall genome. A model on the lesion-specific repair in the context of the molecular defect in TTD is proposed. The implication of the defective CPD repair and efficient 6-4PP repair subpathways in cancer prevention in TTD patients is discussed.

Cell Line↗

PubMed1995

The Cockayne syndrome group A gene encodes a WD repeat protein that interacts with CSB protein and a subunit of RNA polymerase II TFIIH.

The hereditary disease Cockayne syndrome (CS) is characterized by a complex clinical phenotype. CS cells are abnormally sensitive to ultraviolet radiation and are defective in the repair of transcriptionally active genes. The cloned CSB gene encodes a member of a protein family that includes the yeast Snf2 protein, a component of the transcriptional regulator Swi/Snf. We report the cloning of the CSA cDNA, which can encode a WD repeat protein. Mutations in the cDNA have been identified in CS-A cell lines. CSA protein interacts with CSB protein and with p44 protein, a subunit of the human RNA polymerase II transcription factor IIH. These observations suggest that the products of the CSA and CSB genes are involved in transcription.

Amino Acid Sequence↗

PubMed1995

Inflammatory mediators increase surface expression of integrin ligands, adhesion to lymphocytes, and secretion of interleukin 6 in mouse Sertoli cells.

The expression of the cell adhesion molecules ICAM-1, ICAM-2, and VCAM-1 and the secretion of the cytokine interleukin 6 have been measured in mouse Sertoli cells cultured in vitro. Cytometric analysis revealed that, in basal conditions, low levels of ICAM-1 and VCAM-1 were present on the surface of the cells, whereas treatment with interleukin 1, tumor necrosis factor alpha, lipopolysaccharide, or interferon gamma induced, with different kinetics, increases in their expression. ICAM-2 was not detectable in basal conditions, nor was it inducible. Electron microscopic analysis and binding experiments using 51Cr-labeled lymphocytes demonstrated that increased expression of ICAM-1 and VCAM-1 on the surface of Sertoli cells, induced by inflammatory mediators, determines an augmented adhesion between the two cell types. The same stimuli, with the exception of interferon gamma, produced a rapid and remarkable increment of interleukin 6 production by Sertoli cells. These results suggest the presence of both direct and paracrine mechanisms of interaction between Sertoli and immune-competent cells, possibly involved in the control of immune reactions in the testis. Such mechanisms are of interest for the understanding of autoimmune pathologies of the testis and, if confirmed in humans, they could be involved in the sexual transmission of human immunodeficiency virus infection.

Animals↗

PubMed1995

Late induction of human DNA ligase I after UV-C irradiation.

We have studied the regulation of DNA ligase I gene expression in UV-C irradiated human primary fibroblasts. An increase of approximately 6-fold both in DNA ligase I messenger and activity levels was observed 24 h after UV treatment, when nucleotide excision repair (NER) is no longer operating. DNA ligase I induction is serum-independent and is controlled mainly by the steady-state level of its mRNA. The activation is a function of the UV dose and occurs at lower doses in cells showing UV hypersensitivity. No increase in replicative DNA polymerase alpha activity was found, indicating that UV induction of DNA ligase I occurs through a pathway that differs from the one causing activation of the replication machinery. These data suggest that DNA ligase I induction could be linked to the repair of DNA damage not removed by NER.

Cells, Cultured↗

PubMed1995

Development of a new easy complementation assay for DNA repair deficient human syndromes using cloned repair genes.

Nucleotide excision repair (NER)-deficient human cells have been assigned so far to a genetic complementation group by a somatic cell fusion assay and, more recently, by microinjection of cloned DNA repair genes. We describe a new technique, based on the host cell reactivation assay, for the rapid determination of the complementation group of NER-deficient xeroderma pigmentosum (XP), Cockayne's syndrome (CS) and photosensitive trichothiodystrophy (TTD) human cells by cotransfection of a UV-irradiated reporter plasmid with a second vector containing a cloned repair gene. Expression of the reporter gene, either chloramphenicol acetyltransferase (CAT) or luciferase, reflects the DNA repair ability restored by the introduction of the appropriate repair gene. All genetically characterized XP, CS and TTD/XP-D cells tested failed to express the UV-irradiated reporter gene, this reflecting their NER deficiency whereas cotransfection with the repair plasmid expressing a gene specific for the given complementation group increased the enzyme activity to the level reached by normal cells. Selective recovery of both reporter enzyme activities was observed after cotransfection with the XPC gene for the XP17VI cells and with the XPA gene for both XP18VI and XP19VI cells. Using this method, we assigned three new NER-deficient human cells obtained from patients presenting clinical symptoms described as classical XP to either XP group A (XP18VI and XP19VI) and XP group C (XP17VI). Therefore, this technique increases the range of methods now available to determine the complementation group of new NER deficient patients with the advantage, unlike the somatic cell fusion assay or the microinjection procedure, of being simple, rapid, and inexpensive.

Biopsy↗

PubMed1995

Integrin receptor alpha 6 beta 1 is localized at specific sites of cell-to-cell contact in rat seminiferous epithelium.

With the aim of investigating the presence and role of integrin receptors in cell-to-cell interactions during spermatogenesis, we have immunolocalized alpha 6A integrin chain in the rat seminiferous epithelium. In both prepubertal and adult seminiferous epithelium, the antigen was found to be restricted to definite sites of intercellular contact, following a stage-specific distribution almost invariably identical to that known for beta 1 chain. In the adult seminiferous epithelium, positivity for both antigens was found exclusively around the profiles of elongating and maturing spermatids and, in most but not all stages, at a characteristic suprabasal position. In the prepubertal rat, the antigen is localized along a very regular suprabasal line of intercellular contacts. In immunoprecipitation experiments on both seminiferous epithelium explants and Sertoli cell cultures from 3-wk-old rats, anti-alpha 6A antibody coprecipitates a polypeptide of 118 kDa, presumably corresponding to beta 1 chain. These data strongly suggest that the integrin heterodimer alpha 6A beta 1 is expressed at sites of intercellular contact in the rat seminiferous epithelium. The stage-specific and restricted pattern observed by immunofluorescence suggests that this integrin is involved in regulatory interactions between Sertoli cells and germ cells during spermatogenesis.

Animals↗

PubMed1995

Activin stimulates Sertoli cell proliferation in a defined period of rat testis development.

The action of activin-A on Sertoli and spermatogonial cell proliferation during early postnatal life was studied by using in vitro organ culture of testis fragments from 9-day-old rats. Activin significantly stimulated 3H-thymidine incorporation into testis fragments cultured for 3 days in the presence of FSH, whereas it had no effect in the absence of the hormone. This effect was dose dependent in the range 10-200 ng/ml and was specifically inhibited by the activin-binding protein, follistatin. The effect of activin upon proliferation of different testicular cells was studied in detail by 5-bromo-2'-deoxyuridine-labeling fragments at the end of in vitro culture and then determining percentages of different labeled cells on immunostained histological sections. Concomitant treatment with FSH and activin, but not with FSH or activin alone, significantly stimulated Sertoli cell proliferation but markedly depressed that of differentiating type A spermatogonia. In contrast, proliferative activity of undifferentiated type A spermatogonia was independent of activin, irrespective of the presence of FSH. The effect of donor animal age was then investigated by culturing fragments derived from 3- and 18-day-old rats for 3 days. An age-related response was evident. Sertoli cell proliferation was stimulated by FSH alone in fragments from 3-day-old rats, activin having no apparent effect at this age. In contrast, none of the hormones tested either alone or in combination was effective in 18-day-old animals. These results demonstrate that activin acts with FSH in maintaining mitotic potentiality of Sertoli cells in a defined phase of their maturation path, when their proliferative activity is approaching the final arrest. These findings suggest that activin may be an important local factor in regulating Sertoli cell number and that the mitosis of differentiating spermatogonia subsides during Sertoli cell proliferation.

Activins↗

PubMed1995

Stage and cell-specific expression of the adenosine 3',5' monophosphate-phosphodiesterase genes in the rat seminiferous epithelium.

Four genes (ratPDE1/IVc, ratPDE2/IVa, ratPDE3/IVd, and ratPDE4/IVb) encoding different isoforms of phosphodiesterase that specifically hydrolyze the second messenger cAMP (cAMP-PDEs) are present in the rat. Previous data from our laboratory indicated that these genes are differentially expressed in the somatic and germ cells of the seminiferous epithelium of the testis. To further characterize their spatial and temporal expression in the seminiferous tubules, in situ hybridization was used to monitor the expression of the four cAMP-PDE messenger RNAs (mRNAs). The signals corresponding to ratPDE1/IVc and ratPDE2IVa mRNAs were localized in two restricted layers of the seminiferous epithelium. The ratPDE1/IVc mRNA was present in a region of the epithelium corresponding to the location of middle-late pachytene spermatocytes. Conversely, the ratPDE2/IVa signal was confined to a more adluminal area corresponding to the location of maturing round spermatids. The ratPDE3/IVd and ratPDE4/IVb mRNAs were distributed throughout the span of the seminiferous epithelium, indicating a localization in the Sertoli cell cytoplasm. Although the intensity of the signal corresponding to ratPDE4/IVb was similar in all seminiferous tubule stages, the ratPDE3/IVd signal varied in intensity in tubules at different stages of the seminiferous cycle. Maximal expression was present in tubules at stages I-V and XI-XIII of the cycle and minimal at stages VIII-IX of the cycle. The expression of the ratPDE3/IVd mRNA positively correlated with the ability of specific inhibitors of the cAMP-PDEs to potentiate the FSH-dependent cAMP accumulation in tubules at different stages of the seminiferous cycle, with maximal potentiation observed at stages II-VI of the cycle. These data demonstrate that different cAMP-PDE genes are active in different cells of the seminiferous tubules and that the ratPDE3/IVd gene is expressed in the Sertoli cell in a cyclical fashion during the seminiferous cycle.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1995

Molecular and cellular analysis of the DNA repair defect in a patient in xeroderma pigmentosum complementation group D who has the clinical features of xeroderma pigmentosum and Cockayne syndrome.

Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are quite distinct genetic disorders that are associated with defects in excision repair of UV-induced DNA damage. A few patients have been described previously with the clinical features of both disorders. In this paper we describe an individual in this category who has unusual cellular responses to UV light. We show that his cultured fibroblasts and lymphocytes are extremely sensitive to irradiation with UV-C, despite a level of nucleotide excision repair that is 30%-40% that of normal cells. The deficiency is assigned to the XP-D complementation group, and we have identified two causative mutations in the XPD gene: a gly-->arg change at amino acid 675 in the allele inherited from the patient's mother and a -1 frameshift at amino acid 669 in the allele inherited from his father. These mutations are in the C-terminal 20% of the 760-amino-acid XPD protein, in a region where we have recently identified several mutations in patients with trichothiodystrophy.

Cells, Cultured↗

PubMed1995

The product of Zfp59 (Mfg2), a mouse gene expressed at the spermatid stage of spermatogenesis, accumulates in spermatozoa nuclei.

We have described previously three mouse multifinger coding genes (C. Passananti et al., Proc. Natl. Acad. Sci. USA, 86: 9417-9421, 1989). We have analyzed the expression of one of them, termed Mfg2 and renamed Zfp59, and demonstrated that Zfp59 mRNA and its translation product are present in specific stages of mouse spermatogenesis. The predicted amino acid sequence of the Zfp59 protein has been found to consist of 16 zinc-finger motifs clustered at the COOH terminus and subdivided into two groups by a degenerate finger motif. At its NH2 terminus, Zfp59 shares with other members of the zinc finger gene family two additional conserved amino acid modules A and B, described as either FAX, KRAB, or FPB domains. By means of Northern blot, Western blot analysis, and immunohistochemical localization, Zfp59 mRNA and its translation product were shown to be synthesized specifically during the postmeiotic phase of male germ line differentiation. By immunoelectron microscopy, the Zfp59 protein has been shown to accumulate in the nuclei of mature sperms in association with the nuclear matrix.

Amino Acid Sequence↗

PubMed1994

Molecular analysis of the XP-D gene in Italian families with patients affected by trichothiodystrophy and xeroderma pigmentosum group D.

In several patients with the rare hereditary disorder trichothiodystrophy (TTD), a DNA repair defect has been shown to be in the same gene as in xeroderma pigmentosum complementation group D (XP-D). The ERCC-2 gene (excision repair cross-complementing rodent repair deficiency of group 2) has recently been identified as a strong candidate gene for XP-D, since it restores normal UV sensitivity to XP-D cells after transfection. Using Southern blotting, we have analysed the ERCC-2 gene in DNA samples from 28 members of nine Italian families with individuals affected by XP-D (three patients) or by TTD with photosensitivity due to the XP-D defect (eight patients). No major modifications of the ERCC-2 gene were detected with two cDNA probes in either XP-D or TTD patients indicating that the association between TTD and XP-D is not likely to result from a large deletion or rearrangement involving this gene. We found two RFLPs after digestion of the DNA samples with TaqI or MspI, but neither of them could be related to the molecular alteration determining the pathological phenotype. We also analysed a human homologue detected with the hamster sequence isolated by Arrand et al. (1989), which specifically, but partially, complements the DNA repair deficiency in XP-D cells. Our analysis demonstrated that this gene is not the primary gene defective in XP-D. In fact two RFLPs detected with a genomic probe do not co-segregate with the disease in an XP-D family.

DNA↗