Differential repair of UV damage in rad mutants of Saccharomyces cerevisiae: a possible function of G2 arrest upon UV irradiation.

After UV irradiation, the transcriptionally active MAT alpha locus in Saccharomyces cerevisiae is preferentially repaired compared with the inactive HML alpha locus. The effect of rad mutations from three different epistasis groups on differential repair was investigated. Three mutants, rad9, rad16, and rad24, were impaired in the removal of UV dimers from the inactive HML alpha locus, whereas they had generally normal repair of the active MAT alpha locus. Since RAD9 is necessary for G2 arrest after UV irradiation, we propose that the G2 stage plays a role in making the dimers accessible for repair, at least in the repressed HML alpha locus.

Inheritance of abnormal expression of SOS-like response in xeroderma pigmentosum and hereditary cancer-prone syndromes.

The time course of induction of SOS-like stress responses such as enhanced reactivation (ER) and enhanced mutagenesis (EM) has been investigated in UV-C-irradiated skin fibroblasts from a xeroderma pigmentosum (XP) family, using herpes simplex virus type 1 as a probe. Similar ER studies were performed in a Li-Fraumeni syndrome (LFS) family and in a family with a high incidence of breast, ovarian, and colon cancer. In two XP (complementation group B) patients, with a striking absence of skin tumors even at an age of >40 years, only induction of EM was observed, whereas ER was absent (XPER-). The ER- phenotype was inherited from the father, whereas cells from the mother exhibited normal expression of ER and EM. This suggests that the absence of ER is a hereditary trait that is not correlated with a repair-deficient phenotype. Abnormally high levels of ER were observed in UV-C-exposed skin fibroblasts from rive LFS patients. The inheritance of the ER response was studied in one LFS family. High levels of ER were observed only in cells derived from affected individuals carrying one mutated p53 allele, whereas cells from unaffected family members, carrying two wild-type p53 alleles, exhibited normal ER levels. This result shows that abnormally high levels of ER positively correlate with the occurrence of cancer in affected individuals from a LFS family. Interestingly, abnormally high levels of ER were observed in cells from afflicted as well as from unafflicted members of a family with a high incidence of breast, ovarian, colon, and stomach cancer. This suggests that these latter individuals have inherited a mutated, putative predisposing gene, resulting in abnormal expression of ER, but that cancer had not yet developed. The results indicate that the ER response can possibly be used as a prognostic marker to identify carriers in various hereditary cancer-prone syndromes at an early age.

A lack of radiation-induced ornithine decarboxylase activity prevents enhanced reactivation of herpes simplex virus and is linked to non-cancer proneness in xeroderma pigmentosum patients.

Patients with xeroderma pigmentosum (XP), a DNA repair disorder, run a large risk of developing skin cancer in sun-exposed areas. Cancer proneness in these patients correlates with a mammalian SOS-like response, "enhanced reactivation (ER) of viruses." Here, we report that radiation-induced activation of the ornithine decarboxylase (ODC) gene, a putative proto-oncogene, is required for this response. Various diploid fibroblast strains derived from a non-cancer-prone subclass of XP patients, which lack the ER response, were irradiated with 2 J/m2 and assessed for gene induction. In these fibroblasts, an absence of induction of ODC by UV-C was observed at the levels of mRNA, protein, and enzyme activity. This lack of induction is quite specific because the genes for fos and collagenase were induced as they were in normal XP cells. The apparent linkage between non-cancer proneness and a lack of ER and ODC induction was confirmed in a fibroblast strain derived from a patient with another DNA repair disorder, trichothiodystrophy, which does not lead to cancer proneness: in these cells, no induction of the ER response nor of ODC occurs after UV-C irradiation. Repair deficiency, however, is not essential because the simultaneous lack of ODC and ER induction after 10 J/m2 UV-C was found in at least one repair-proficient fibroblast. Next, a specific inhibitor of ODC, difluoromethylornithine, at a dose of 10 mM, completely blocked the ER response in cultured normal skin fibroblasts, suggesting that the ODC enzyme is in fact essential for the ER response. Difluoromethylornithine, although it did not affect other processes such as DNA repair, leads to a block in the cell division cycle at the G1-S transition. Interestingly, other blockers of this transition, wortmannin (500 nM) and mimosine (100 mM), also decreased the ER response. Finally, the ER and ODC responses also seem to be linked after treatment with X-irradiation (3 Gy), suggesting that both are part of a general response to DNA damage, at least in human skin fibroblasts. Apart from the abnormal ER and ODC responses, fibroblasts from non-tumor-prone XP patients react in the same way to radiation as do fibroblasts from tumor-prone XP patients with respect to other parameters. Thus, the lack of ODC induction after radiation may help to protect XP patients against skin carcinogenesis.

Bloom's syndrome cells GM1492 lack detectable p53 protein but exhibit normal G1 cell-cycle arrest after UV irradiation.

The tumor suppressor gene p53 is thought to be a key factor in the onset of G1 cell-cycle arrest following DNA damage. However, here we describe cells derived from a patient with Bloom's syndrome, lacking any detectable p53 protein, that still shows a functional G1 cell-cycle checkpoint after irradiation with UV-C. Comparison with cells from other Bloom's patients showed that the absence of p53 protein is not a specific characteristic of Bloom's syndrome.

Mif1: a missing link between the unfolded protein response pathway and ER-associated protein degradation?

Eukaryotic cells have three different mechanisms to deal with the accumulation of unfolded proteins in the endoplasmic reticulum: (1) In cells in which unfolded polypeptides accumulate, translation initiation is inhibited to prevent further accumulation of unfolded proteins. (2) Expression of proteins involved in polypeptide folding is strongly enhanced by a process called the Unfolded Protein Response (UPR). (3) Proteins missing the proper tertiary structure are degraded by the ER-Associated protein Degradation (ERAD) mechanism. Recent studies in S. cerevisiae have shown that the processes of UPR and ERAD are functionally linked to each other. Cells lacking a functional ERAD show a constitutive activation of UPR. In addition, many of the components of ERAD are under the direct transcriptional control of UPR. Finally, while neither UPR nor ERAD are essential for cell viability, deletion of both pathways results in severe growth impairment. UPR and ERAD are conserved between yeast and mammalian cells. One of the components of mammalian UPR is the protease presenilin-1. Mutations in the gene for presenilin-1 cause early-onset familial Alzheimer disease. Interestingly, inhibition of proteolysis by the ubiquitin-26S proteasome system has also been described for Alzheimer s disease. This suggests a link between UPR and ERAD in mammalian cells. The recently identified gene Mif1 is a possible candidate to form a direct link between UPR and ERAD in mammalian cells. The Mif1 gene is under the direct control of UPR. Mif1 is a trans-ER-membrane protein, with both the N- and the C-termini facing the cytoplasmic side of the ER membrane. It contains an N-terminal ubiquitin-like domain. It is anticipated that Mif1 may associate through its ubiquitin-like domain with the 26S proteasome, in this way connecting the protein degradation machinery to the ER membrane and resulting in an efficient ERAD.

The novel MMS-inducible gene Mif1/KIAA0025 is a target of the unfolded protein response pathway.

In a search for genes induced by DNA-damaging agents, we identified two genes that are activated by methyl methanesulfonate (MMS). Expression of both genes is regulated after endoplasmic reticulum (ER) stress via the unfolded protein response (UPR) pathway. The first gene of those identified is the molecular chaperone BiP/GRP78. The second gene, Mif1, is identical to the anonymous cDNA KIAA0025. Treatment with the glycosylation inhibitor tunicamycin both enhances the synthesis of Mif1 mRNA and protein. The Mif1 5' flanking region contains a functional ER stress-responsive element which is sufficient for induction by tunicamycin. MMS, on the other hand, activates Mif1 via an UPR-independent pathway. The gene encodes a 52 kDa protein with homology to the human DNA repair protein HHR23A and contains an ubiquitin-like domain. Overexpressed Mif1 protein is localized in the ER.

Different regulation of p53 stability in UV-irradiated normal and DNA repair deficient human cells.

The stabilization of p53 protein was studied after UV exposure of normal human skin fibroblasts and cells derived from patients suffering from xeroderma pigmentosum (XP) and trichothiodystrophy (TTD). The data show that p53 is transiently stabilized both in UV-irradiated normal and repair deficient cells. However, particularly at later times after UV irradiation, stabilization of p53 persists much longer in repair deficient XP and TTD cells than in normal cells. The stabilization of p53 was found to be dose-dependent in normal and XP cells. These results indicate that unremoved DNA damage could possibly be responsible for the induction of transient stabilization of p53.

Impaired DNA repair capacity in skin fibroblasts from various hereditary cancer-prone syndromes.

Host-cell reactivation (HCR) of UV-C-irradiated herpes simplex virus type 1 (HSV-1) has been determined in skin fibroblasts from the following hereditary cancer-prone syndromes: aniridia (AN), dysplastic nevus syndrome (DNS), Von Hippel-Lindau syndrome (VHL), Li-Fraumeni syndrome (LFS) and a family with high incidence of breast and ovarian cancer. Cells from AN, DNS or VHL patients were found to exhibit heterogeneity in HCR. Cells from individuals belonging to an LFS family show reduced HCR in all cases where the cells were derived from persons carrying one mutated p53 allele, whereas cells derived from members with two wild-type alleles show normal HCR. LFS cells with reduced HCR also reveal reduced genome overall repair, and a slower gene-specific repair of the active adenosine deaminase (ADA) gene, but little if any repair of the inactive 754 gene. In the breast/ovarian cancer family, reduced HCR is observed in skin fibroblasts derived from both afflicted and unaffected individuals. In addition, these cells display lower survival after exposure to UV-C and exhibit higher levels of SCEs than those in normal cells. These observations indicate that various hereditary cancer-prone syndromes, carrying mutations in different tumor-suppressor genes, exhibit an unexplained impairment of the capacity to repair UV-damaged DNA.

Down-regulation of T-STAR, a growth inhibitory protein, after SV40-mediated immortalization.

Normal human cells can undergo a limited number of divisions, whereas transformed cells may have an extended life span and can give rise to immortal cells. To isolate genes involved in the immortalization process, gene expression in SV40-transformed preimmortal human fibroblasts was compared with expression in SV40-transformed immortalized fibroblasts using an mRNA differential display. We found that the growth-inhibitory protein testis-signal transduction and activation of RNA (T-STAR) a homologue of cell-cycle regulator Sam68, is strongly down-regulated in immortalized cells. Overexpression of T-STAR in the SV40-transformed immortalized cells resulted in a strong reduction of colony formation, whereas deletion of the RNA-binding domain of T-STAR abrogated this effect. Down-regulation of testis-signal transduction and activation of RNA (T-STAR) expression is found only in immortal cells isolated after a proliferative crisis accompanied with massive cell death. The strict correlation of down-regulation of T-STAR expression only in those immortal cells that arose after a clear proliferative crisis suggests that the loss of T-STAR might be necessary to bypass crisis.

Induction of the SAPK activator MIG-6 by the alkylating agent methyl methanesulfonate.

The alkylating agent methylmethanesulfonate (MMS) activates the c-jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) and the p38 mitogen-activated protein kinase (p38MAPK) pathways via different mechanisms of action. Activation of p38MAPK by MMS involves the pp125 focal adhesion kinase-related tyrosine kinase RAFTK and the MAPK kinase 3. The way in which MMS can activate JNK/SAPK has not been elucidated. Here we describe the identification by differential display of human mitogen-activated gene-6 (MIG-6) as a novel MMS-inducible gene. Induction of MIG-6 by MMS was found in human diploid skin fibroblasts and in simian virus 40-transformed skin fibroblasts, indicating that the enhanced expression of MIG-6 after MMS-treatment did not require p53. The signal leading to activation of MIG-6 appeared to be independent of DNA damage. High MIG-6 expression was found in the liver, lung, and placenta. MIG-6 is an adapter protein that binds to the activated form of cdc42Hs and to 14-3-3 proteins, thereby activating JNK/SAPKs. Our results suggest that activation of JNK/SAPKs by MMS may involve the induction of MIG-6.

Differential repair of UV damage in Saccharomyces cerevisiae is cell cycle dependent.

In the yeast Saccharomyces cerevisiae the transcriptionally active MAT alpha locus is repaired preferentially to the inactive HML alpha locus after UV irradiation. Here we analysed the repair of both loci after irradiating yeast cells at different stages of the mitotic cell cycle. In all stages repair of the active MAT alpha locus occurs at a rate of 30% removal of dimers per hour after a UV dose of 60 J/m2. The inactive HML alpha is repaired as efficiently as MAT alpha following irradiation in G2 whereas repair of HML alpha is less efficient in the other stages. Thus differential repair is observed in G1 and S but not in G2. Apparently, in G2 a chromatin structure exists in which repair does not discriminate between transcriptionally active and inactive DNA or, alternatively, an additional repair mechanism might exist which is only operational during G2.

Differential repair of UV damage in Saccharomyces cerevisiae.

Preferential repair of UV-induced damage is a phenomenon by which mammalian cells might enhance their survival. This paper presents the first evidence that preferential repair occurs in the lower eukaryote Saccharomyces cerevisiae. Moreover an unique approach is reported to compare identical sequences present on the same chromosome and only differing in expression. We determined the removal of pyrimidine dimers from two identical alpha-mating type loci and we were able to show that the active MAT alpha locus is repaired preferentially to the inactive HML alpha locus. In a sir-3 mutant, in which both loci are active this preference is not observed.

GM1492 human diploid skin fibroblasts lack the p53-dependent G1 cell-cycle checkpoint.

Recently, we reported that GM1492 human diploid skin fibroblasts derived from a Bloom's patient upon UV-C irradiation fail to increase p53 to a detectable level and nevertheless accumulate in the G1-phase of the cell-cycle. Here we show that in GM1492 cells other types of DNA-damaging agents also fail to induce p53 as well as WAF1, a p53-regulated gene product involved in G1 cell-cycle arrest. Furthermore, the p53-dependent G1 cell-cycle checkpoint is indeed defective in these cells: However, induction of GADD45 mRNA still occurs in GM1492 after irradiation with UV-C. Since GADD45 is known to inhibit the entry into S, these data suggest that the observed accumulation of GM1492 cells in G1 after UV-C irradiation occurs at the G1/S boundary and is due to an inhibition of initiation of DNA-replication.

Distinct modulation of p53 activity in transcription and cell-cycle regulation by the large (54 kDa) and small (21 kDa) adenovirus E1B proteins.

P53 can both stimulate transcription via the p53-consensus sequence as well as inhibit gene expression via CAAT-TATA-sequences. Certain viral and cellular proteins can abrogate the p53-dependent stimulation of transcription by physical association. In addition, it has been shown that the large E1B protein of adenovirus type 12 (Ad12), E1B/54 kDa, can block the transcription activation potential of p53, without binding to p53. Here we show that this E1B/54-kDa protein also can prevent the repression of transcription of transfected and endogenous p53 in transient transfections. In cells containing wild-type p53 but stably expressing high levels of E1B/54 kDa, no induction of WAF1 mRNA after X-ray irradiation could be detected. In contrast, expression of another non-p53 binding E1B protein, Ad5 E1B/21 kDa has no effect on WAF-1 expression. Results of an electromobility shift assay indicated that the abrogation of p53-mediated transcription activation by E1B/54 kDa cannot be explained by inhibition of the DNA-binding capacity of p53. A biological consequence of expression of E1B/54 kDa is the loss of G1 cell-cycle arrest after X-ray irradiation, while cells expressing the E1B/21 kDa still arrest in G1 after DNA damage.

Temperature-sensitive mutant p53 (ala143) interferes transiently with DNA-synthesis and cell-cycle progression in Saos-2 cells.

It has been demonstrated that temperature-sensitive mutant p53 (val-->ala143) inhibits cell-proliferation at the permissive temperature, albeit to a lesser extent than wild-type p53 (Zhang et al.: EMBO J 13:2535-2544, 1994). We have studied its effect on the cell-cycle by dual-parameter flow cytometry, extended pulse-labeling, and pulse-chase experiments. p53ala143 interferes in Saos-2 cells at three levels with cell-cycle progression at permissive temperatures: it caused a G1-arrest, a reduced rate of DNA synthesis during S, and a prolonged G2/M. Strikingly, all these effects are transient. Continued culturing at 32 degrees C resulted in normal cell-cycle progression. Abrogation of the G1-block occurred even in the presence of high p21Waf1 protein levels, a negative cell-cycle regulator of which the expression is induced by wild-type p53.

Production of fimbrial adhesins K99 and F41 by enterotoxigenic Escherichia coli as a function of growth-rate domain.

The production of fimbrial adhesins K99 and F41 by enterotoxigenic Escherichia coli has been measured in steady-state chemostat experiments at various specific growth rates (microseconds) and in a recycling fermentor across a range of mu values falling to less than 0.004 h-1. It has been demonstrated that the production of K99 and F41 fimbriae is correlated with mu both in aerobic and anaerobic chemostat experiments. A significant production of fimbriae was only detected at mu values higher than 0.2 h-1. This behavior was further examined by culturing the bacteria in a recycling fermentor with complete biomass retention. It could be shown that the production of K99 and F41 fimbriae only occurred during balanced growth, with a high biomass yield at mu values higher than 0.04 h-1 corresponding to mass doubling times (td) of less than 17 h. The production of both fimbriae halted during balanced growth with a lower biomass yield (at mu values between 0.012 and 0.04 h-1 corresponding to td values between 17 and 58 h) and unbalanced stringent growth (at mu values lower than 0.012 h-1 or td values higher than 58 h). The external pH of the medium greatly influenced the production of both K99 and F41 fimbriae. At pH values lower than 7, the production of fimbriae was strongly inhibited. Also, at pH values higher than 7, a decrease in production was observed. The consequences of the observed phenomena for the pathogenic behavior of this E. coli strain are discussed.