Biomedical subjects
M Yamaizumi
Publications and source records attributed to M Yamaizumi.
[Cockayne syndrome].
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Heat shock induces transient p53-dependent cell cycle arrest at G1/S.
Heat shock (43 degrees C, 45 min) induced transient nuclear accumulation of p53 in primary human fibroblasts without any clonogenically toxic effects. The accumulation of p53 reached a maximal level 3 approximately 5 h after heat shock, and returned to the basal level within 12 h. Following the increase in p53 level, cell cycle arrest at G1/S was observed in normal fibroblasts, whereas neither nuclear accumulation of p53 nor cell cycle arrest were observed in HeLa cells. By comparing cell cycle patterns of heat-treated mouse cells with different genotypes at the p53 locus (+/+, +/-, -/-), the observed cell cycle arrest at G1/S was demonstrated to be p53-dependent. Cell cycle arrest in normal human fibroblasts continued for nearly 24 h, resulting in a one day delay of cell growth compared with non-treated cells. Following enhancement of the p53 level, the amount of p21/WAF1/ CIP1 increased, and the high level of p21 was sustained for almost one day in a cell cycle-independent manner, suggesting the involvement of p21 in the inhibition of cell cycle progression by heat shock.
Visualization of mitochondrial protein import in cultured mammalian cells with green fluorescent protein and effects of overexpression of the human import receptor Tom20.
The presequence of the ornithine transcarbamylase precursor (pOTC) was fused to green fluorescent protein (GFP), yielding pOTC-GFP and pOTCN-GFP containing the presequence plus 4 and 58 residues of mature ornithine transcarbamylase, respectively. When GFP cDNA was transfected into COS-7 cells, the cytosol and nucleus were fluorescent. On the other hand, pOTC-GFP cDNA gave strong fluorescence of a unique mitochondrial pattern. After fractionation of cells expressing pOTC-GFP with digitonin, fluorescence was recovered mostly in the particulate fraction. Immunoblot analysis showed that processed GFP was present in the particulate fraction, whereas pOTC-GFP was recovered in both the soluble and particulate fractions. pOTC-GFP and pOTCN-GFP synthesized in vitro were imported efficiently into the isolated mitochondria. Single and triple amino acid mutations in the presequence resulted in impaired mitochondrial import and in a loss of mitochondrial fluorescence. Perinuclear aggregation of fluorescent mitochondria was observed when the human mitochondrial import receptor Tom20 (hTom20) was coexpressed with pOTC-GFP. Overexpression of hTom20 (not DeltahTom20, which lacks the anchor sequence) resulted in stimulated mitochondrial import of pOTC-GFP in COS-7 cells. When pOTC-GFP cDNA was microinjected into nuclei of human fibroblast cells, mitochondrial fluorescence was detected as early as 2-3 h after injection. These results show that GFP fusion protein can be used to visualize mitochondrial structures and to monitor mitochondrial protein import in a single cell in real time.
Cell cycle control is aberrant in Chinese hamster ovary cell mutants exhibiting apoptosis after serum deprivation.
We isolated mutants of Chinese hamster ovary cells that exhibit excessive apoptosis after serum deprivation. In the medium containing 10% serum, the growth rates of the mutants were 1.4 to 1.5-fold faster than those of wild-type cells. Whereas the cell cycle of wild-type cells was arrested at the G1 phase after serum deprivation, the cell cycle of the mutant cells was not fully arrested at this phase, suggesting that cell cycle regulation was disorganized in the mutants. The mutants were highly sensitive to a nucleotide-analogue 5-fluorouracil in the absence of serum, whereas wild-type cells were resistant to the drug. Based on the sensitivity to the drug after serum deprivation, we could classify the mutants into dominant groups and at least two recessive complementation groups. Thus, these mutants presumably contain different lesions in gene(s) required for cell cycle regulation and apoptosis.
[Induction of the p53 response].
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Roles of XPG and XPF/ERCC1 endonucleases in UV-induced immunostaining of PCNA in fibroblasts.
To investigate the relationship between proliferating cell nuclear antigen (PCNA) complex formation and dual incisions in the nucleotide excision repair (NER) process, xeroderma pigmentosum group G (XP-G), XP-F, and XP-G equivalent mouse UV-sensitive mutant ERCC group 5 cells were utilized as a model in this study. These cells are deficient in endonucleases related to 3' (XP-G and ERCC group 5) or 5' (XP-F) incision of the DNA lesions in the NER process. PCNA complex formation was detected by an indirect immunofluorescence method after the cells were fixed in methanol. When Sps1 (XP-G) and XL216-7 (ERCC group 5) cells were UV irradiated, neither of them showed PCNA staining. In contrast, SFN4 (a human normal strain) and heterokaryons of Sps1 and XP96TO (XP-A) cells fused by polyethylene glycol treatment showed PCNA staining following UV irradiation. Furthermore, XLgfPAneo1 cells, derived from XL216-7 cells transfected with a plasmid containing mouse ERCC5 (xpg) cDNA, also restored staining and UV sensitivity. On the other hand, we observed a very faint PCNA staining in XP2YO (XP-F) cells, expressing no detectable ERCC1 or XPF protein, after UV irradiation. X rays induced PCNA staining in all cell lines with a similar staining pattern, and radiosensitivity was exactly the same between XL216-7 and XLgfPAneo1 cells. These results may have implications for the NER process in vivo in that coordinately occurring dual incisions by XPG and XPF/ERCC1 proteins play an important role in inducing PCNA complex formation, but the step may not be required for PCNA-dependent repair of X-ray-induced DNA damage.
Rodent complementation group 8 (ERCC8) corresponds to Cockayne syndrome complementation group A.
US31 is a UV-sensitive mutant cell line (rodent complementation group 8) derived from a mouse T cell line L5178Y. We analyzed removal kinetics for UV-induced cyclobutane pyrimidine dimers and (6-4) photoproducts in US31 cells using monoclonal antibodies against these photoproducts. While nearly all (6-4) photoproducts were repaired within 6 h after UV-irradiation, more than 70% of cyclobutane pyrimidine dimers remained unrepaired even 24 h after UV-irradiation. These kinetics resembled those of Cockayne syndrome (CS) cells. Since US31 cells had a low efficiency of cell fusion and transfection, which hampered both complementation tests and gene cloning, we constructed fibroblastic complementation group 8 cell line 6L1030 by fusion of US31 cells with X-irradiated normal mouse fibroblastic LTA cells. Complementation tests by cell fusion and transfection using 6L1030 cells revealed that rodent complementation group 8 corresponded to CS complementation group A.
Cockayne syndrome complementation group B associated with xeroderma pigmentosum phenotype.
Two siblings have been reported whose clinical manifestations (cutaneous photosensitivity and central nervous system dysfunction) are strongly reminiscent of the DeSanctis-Cacchione syndrome (DCS) variant of xeroderma pigmentosum (XP), a severe form of XP. Fibroblasts from the siblings showed UV sensitivity, a failure of recovery of RNA synthesis (RRS) after UV-irradiation, and a normal level of unscheduled DNA synthesis (UDS), which were, unexpectedly, the biochemical characteristics usually associated with Cockayne syndrome (CS). However, no complementation group assignment in these cells has yet been performed. We here report that these patients can be assigned to CS complementation group B (CSB) by cell fusion complementation analysis. To our knowledge, these are the first patients with defects in the CSB gene to be associated with an XP phenotype. The results imply that the gene product from the CSB gene must interact with the gene products involved in excision repair and associated with XP.
A simple method for diagnosing xeroderma pigmentosum variant.
Patients with xeroderma pigmentosum variant have been diagnosed based on a post-replication repair assay using their cells together with their clinical manifestations. We present here an alternative simple method for the diagnosis of xeroderma pigmentosum variant that measures three cellular markers for DNA repair by autoradiography, unscheduled DNA synthesis, recovery of RNA synthesis, and recovery of replicative DNA synthesis after ultraviolet irradiation. Fibroblasts from a patient are plated on three coverslips parallel with normal cells (control). Unscheduled DNA synthesis, recovery of RNA synthesis, and recovery of replicative DNA synthesis after ultraviolet irradiation in the patient's cells are compared with those of adjacent normal cells by counting numbers of grains on nuclei for each coverslip. Of the hereditary photosensitive disorders including xeroderma pigmentosum, Cockayne syndrome, and newly established ultraviolet-sensitive syndrome, only xeroderma pigmentosum variant cells exhibit normal unscheduled DNA synthesis, normal recovery of RNA synthesis, but reduced recovery of replicative DNA synthesis (approximately 50% of that of control cells). This reduction of DNA synthesis is enhanced in the presence of caffeine. Because each disorder yields a different combination of these three markers, this method also provides a systematic basis for the diagnosis of these diseases.
Clinical characteristics of three patients with UVs syndrome, a photosensitive disorder with defective DNA repair.
Recently, we established a new category of photosensitive disorder termed UVs syndrome. Cells from patients with UVs syndrome have a similar UV sensitivity as xeroderma pigmentosum (XP) cells, but have a normal level of unscheduled DNA synthesis (UDS) unlike XP. UVs syndrome is distinct from Cockayne syndrome (CS) or XP including XP variant (XP-V), as determined by studies of genetic factors using cell fusion, microinjection, and postreplication repair assays. In this study, we identified three Japanese patients with UVs syndrome: an 11-year-old girl, a 17-year-old male, and an 8-year-old boy. The first two patients were siblings, while the third was a case from a different family. All of these patients exhibited acute recurrent sunburn. Common clinical manifestations of these patients were slight erythema and dryness, a number of freckles on sun-exposed areas, and slight telangiectasia only seen on the cheek and nose. Patient 3 showed a lowered minimal erythema dose between 280 and 300 nm. The patients' fibroblasts showed similar characteristics to those in CS, such as UV sensitivity, and a failure of RNA synthesis (RRS) after UV irradiation, despite a normal level of UDS. Thus, UVs syndrome is a new hereditary photosensitive disorder with clinical manifestations similar to a mild form of XP but showing the cellular characteristics of CS.
Separation of protein factors that correct the defects in the seven complementation groups of xeroderma pigmentosum cells.
We fractionated HeLa cell extracts by gel filtration and then micro-injected them into cells derived from the seven complementation groups (A-G) of xeroderma pigmentosum (XP). Distinct fractions that corrected the unscheduled DNA synthesis (UDS) of the complementation group XP cells were identified. The apparent molecular weights corresponding to complementation groups A, B, C, D, E, F, and G were estimated to be 80, 600, 600, 240, 100, 240, and 280 kDa, respectively. These factors were stable in the respective cell lines, the shortest half life being 16 h for the XP-A and XP-G complementing factors. The fraction (80 kDa) that corrected the UDS in XP-A cells also complemented the defect of the XP-A cell extract in the incision of DNA containing a pyrimidine dimer in a cell-free system. The separated fractions will be useful for understanding the molecular nature of these factors and for assigning complementation groups of cells derived from suspected XP patients.
Group D xeroderma pigmentosum: a case with a number of nevocellular nevi.
A 12-year-old boy was diagnosed as having group D xeroderma pigmentosum based on the results of unscheduled DNA synthesis (UDS) tests and complementation tests. More than 200 moles were distributed all over his body, including the unexposed areas of his torso and scalp. All of eight removed specimens were compatible histologically with nevocellular nevi.
Nuclear accumulation of p53 in normal human fibroblasts is induced by various cellular stresses which evoke the heat shock response, independently of the cell cycle.
Nuclear accumulation of p53 is induced by various DNA damaging agents (the p53 response). Induction of nuclear accumulation of p53 after various cellular stresses, mostly other than DNA damage, including heat shock, was examined in normal human fibroblasts by immunostaining and flow cytometry using a mouse anti-p53 monoclonal antibody. Immunostaining revealed nuclear accumulation of p53 within 6 h after various stresses [heat shock, osmotic shock, heavy metal (Cd), blockers of the cellular respiratory system (NaN3), amino acid analogues (azetidine and canavanine), an inhibitor of protein synthesis (puromycin), and oxygen free radicals (H2O2)]. Heat shock proved to be one of the most effective inducers among these stresses. FACScan analysis revealed that this induction of p53 occurred regardless of the stage in the cell cycle and that accumulation of cells in G2/M occurred. As all of these stresses are known to induce the heat shock response, the mechanism of p53 induction after stresses and that of heat shock response may share, at least partly, some common signaling pathway(s).
A young woman with xeroderma pigmentosum complementation group F and a morphoeic basal cell carcinoma.
We report an 18-year-old Japanese woman who had mild photosensitivity and a facial tumour, which was shown to be a morphoeic basal cell carcinoma. Although a line of fibroblasts derived from the patient, Kps6 cells, were slightly more sensitive to UV irradiation than normal cells, their level of unscheduled DNA synthesis was about 20% that of normal cells, and recovery of RNA synthesis after UV irradiation was moderately depressed. Complementation tests, carried out by cell fusion or by microinjection of plasmids harbouring xeroderma pigmentosum (XP) genes, indicated that the patient had XP group F. To our knowledge, this is the youngest XP group F patient with a malignant tumour reported to date.
UVs syndrome, a new general category of photosensitive disorder with defective DNA repair, is distinct from xeroderma pigmentosum variant and rodent complementation group I.
Previously, we reported two DNA repair-defective siblings who did not belong to any complementation group of xeroderma pigmentosum (XP) or Cockayne syndrome (CS). By surveying other photosensitive patients whose fibroblasts showed similar biochemical phenotypes, we found another nonconsanguineous Japanese patient belonging to the same complementation group as our previous cases. Postreplication repair of the cells derived from these patients was normal, indicating that they cannot be classified as XP variant. Neither transfection nor microinjection of the cells with the human DNA repair gene ERCC1, which is known not to correct any complementation groups of XP or CS, failed to correct the defect of these cells, indicating that they do not belong to the rodent complementation group 1. However, the defect in recovery of RNA synthesis (RRS) after UV irradiation was restored by microinjection of HeLa cell extract. Although clinical manifestations of these patients--such as acute sunburn, dryness, freckling, pigmentation anomalies on sun-exposed skin, and teleangiectasia without neurological abnormalities or tumors--are similar to a mild XP phenotype, cellular characteristics such as UV sensitivity and defective RRS after UV irradiation with normal unscheduled DNA synthesis (UDS) are reminiscent of CS. On the basis of these results, we propose that these patients be included under a general category designated "UV-sensitive" (UVs) syndrome.
[The conditions that induce tumor-suppressor protein p53].
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Complementation of xeroderma pigmentosum cells by microinjection of mRNA fractionated under denaturing conditions: an estimation of sizes of XP-E and XP-G mRNA.
Excision repair deficiencies in groups A and G xeroderma pigmentosum (XP) cells are transiently complemented after microinjection of HeLa poly(A)+RNA, but those in groups D and F are not complemented (Legerski et al., 1984). We tested XP cells belonging to the seven complementation groups, A-G, and Cockayne's syndrome (CS) cells belonging to the two complementation groups, A and B, for transient correction by microinjection of total poly(A)+RNA from HeLa cells. Among the XP cells, unscheduled DNA synthesis (UDS) was increased only in XP-A cells by microinjection of total poly(A)+RNA. However, UDS was increased in XP-E and XP-G cells as well as in XP-A cells by microinjection of concentrated poly(A)+RNA fractionated on a 5-25% sucrose density gradient containing methylmercuric hydroxide. The sizes of XP-E and XP-G mRNA were estimated to be 1.5-2.7 kb and 2.0-3.8 kb, respectively, by comparison to internal marker RNAs including 18S rRNA, 28S rRNA, HPRT mRNA and XPAC mRNA. RNA synthesis recovery after UV exposure in CS cells was not increased by microinjection of either total poly(A)+RNA or fractionated RNA. These results provide estimates of the sizes of XP-E and XP-G proteins and will facilitate molecular cloning of DNA repair genes, especially of XP-E and XP-G genes.