Defective reactivation of ultraviolet light-irradiated herpesvirus by a Bloom's syndrome fibroblast strain.

We have used the technique of host cell reactivation of UV-irradiated herpes simplex virus type 1 as a measure of the repair capacity of three Bloom's syndrome skin fibroblast strains. At low multiplicity of infection (less than 6 x 10(-4) plaque-forming unit/cell), reactivation of the virus by the Bloom's syndrome strains was indistinguishable from that by normal strains. Reactivation at higher multiplicities was measured using an infectious centers assay. At 3 plaque-forming units/cell, survival of UV-irradiated herpes simplex virus was higher in all cell strains as a result of the multiplicity reactivation effect. This effect was, however, much smaller in one Bloom's syndrome strain, GM1492, than in either the normal strains or the other Bloom's syndrome fibroblasts. The defect in GM1492 was manifest only at relatively high multiplicates of infection. Thus, at 0.01 plaque-forming unit/cell, the GM1492 strain appeared normal, using the infectious centers assay. Clonal survival of the UV-irradiated GM1492 fibroblasts was also normal. Caffeine at 4 mM had little effect on either virus or cell survival following UV irradiation. The results indicate that the Bloom's syndrome strain GM1492 may be deficient in one of the cellular functions responsible for the multiplicity reactivation effect. These effects include complementation and recombinational events. Alternatively, the GM1492 strain may have a defective UV repair system which becomes saturated at high levels of damage.     

High levels of the c-myc protein in cell lines of Bloom's syndrome origin

Lymphoblastoid cell lines derived from cancer prone Bloom's Syndrome patients differ from cell lines representative of several other disorders by exhibiting a constitutive elevation in the level of the c-myc protein. This may be a contributing factor in the strong predisposition to malignancy observed in Bloom's syndrome.     

Altered formation of DNA replication intermediates in human 46 BR fibroblast cells hypersensitive to 3-aminobenzamide

The appearance of DNA replication intermediates was investigatedin a human fibroblast strain (46 BR) which is hypersensitiveto the lethal effects of 3-aminobenzamide. 3-Aminobenzamideis an inhibitor of poly(ADP-ribose) synthetase and modulatesDNA ligase activity. We detected the same intermediates (10kb DNA and Okazaki-fragments) as in normal fibroblasts, butkinetics and amounts of intermediates were altered, either asa result of, or in order to overcome the defect in the cells.     

3. Chromosomal instability in B-lymphoblasotoid cell lines from Werner's and Bloom's syndrome patients

Werner's syndrome (WS) and Bloom's syndrome (BS) are rare autosomal recessive diseases in which the feature of premature aging and the elevated risk of neoplasia may be associated with genomic instability. To cha-racterize the genomic instability of WS and BS, B-lymphoblastoid cell lines (LCLs) from WS and BS patients were cytogenetically analyzed, comparing to those from healthy donors. Although all     

NAD and the synthesis of (ADP-ribose)n in a human cell strain (46BR) hypersensitive to the lethal effects of 3-aminobenzamide

The cell strain 46BR, derived from an immunodeficient individual,is hypersensitive to the lethal effects of DNA-damaging agents,and of 3-aminobenzamide (3AB), the latter being an inhibitorof the enzyme ADP-ribosyltransferase (ADPRT). This hypersensitivityis not found with the non-inhibitory analogue, 3-aminobenzoate.The NAD content of 46BR cells is similar to that of fibroblastsfrom normal human donors, as is the decrease in NAD contentfollowing treatment with dimethylsulphate. Both the activityof ADP-ribosyltransferase and its inhibition by 3AB in permeabiliz-edcells are similar in 46BR and in normal cell strains. High concentrationsof 3AB interfere with purine metabolism in cultured cells. Againthis effect is similar in 46BR and normal cells. Thus thereis no apparent anomaly either in the activity of ADPRT or inthe gross effects of 3AB in 46BR. The sensitivity to 3AB maybe caused by a defect in a specific acceptor for the ADP-ribosesynthesized by ADPRT, or in some as yet undiscovered actionof the inhibitor.     

Comparison of DNA repair protein expression and activities between human fibroblast cell lines with different radiosensitivities

In order to investigate the molecular basis of variation in response to ionising radiation (IR) in radiotherapy patients, we have studied the expression of several genes involved in DNA double-strand break repair pathways in fibroblast cell lines. Ten lines were established from skin biopsies of cancer patients with different normal-tissue reactions to IR, and 3 from a control individual. For all 10 test cell lines, the cellular radiosensitivity was also known. Using Western blots we measured, in non-irradiated cells, the basal expression levels of ATM, Rad1 and Hus1, involved in the control of cellular DNA damage checkpoints, together with DNA-PKcs, Ku70, Ku80; XRCC4, ligaseIV and Rad51, involved in radiation- induced DSB repair. We also analysed the in vitro enzymatic activities, under non-irradiated conditions, of the DNA-PK and XRCC4/ligaseIV complexes. The levels of expression of the different proteins were similar in all the cell lines, but the activities of the DNA-PK and XRCC4/ligaseIV complexes showed some differences. These differences did not correlate with either the normal tissue response of the patient in vivo or with cellular radiation sensitivity in vitro. The activity differences of these enzyme complexes, therefore, do not account for the variation of responses seen between patients.     

人肿瘤细胞系LigaseⅣ基因突变与放射敏感性的关系研究

目的研究人肿瘤细胞DNA-LigaseⅣ基因突变与放射敏感性的关系.方法常规集落形成方法测定人鼻咽鳞癌(CNE)、肺腺癌(SPC-A1)和乳腺腺癌(MCF-7)细胞系不同剂量照射后的细胞存活分数,采用聚合酶链式反应和克隆技术测定3种细胞参与DNA损伤修复的DNA ligaseⅣ基因序列,分析它们的同源性变化和突变对基因产物亲疏水性的影响.结果 3种细胞存活参数比较存在较大差异,CNE细胞较SPC-A1和MCF-7细胞显示出较高的放射敏感性.CNE、SPC-A1和MCF-7细胞LigaseⅣ基因同源性分别为99.95%、99.99%和99.98%,包括碱基转换和颠换突变.部分突变碱基导致氨基酸替代并影响基因产物的亲疏水结构.CNE细胞LigaseⅣp的313aa His→Arg、538aa Gly→Arg、579aa Lys→Arg和585aa Asn→Ser替代与CNE细胞放射敏感性高于SPC-A1和MCF-7细胞有关.结论 LigaseⅣp在非同源性末端连接(NHEJ)途径中起关键和最终连接作用,该基因突变影响肿瘤细胞的双链断裂损伤修复能力和放射敏感性.     

The HuT series of 'carcinogen-transformed' human fibroblast cell lines are derived from the human fibrosarcoma cell line 8387

In 1977 Kakunaga reported the carcinogen-induced transformationof the diploid human fibroblast cell line KD into focus-forming,morphologically altered cells. Cell lines were developed from15 individual foci. These exhibited an infinite lifespan inculture and all those that were tested (7/7) formed malignanttumors(sarcomas) in athymic mice. The existing cell lines, designatedHuT-11 to HuT-14, have been studied intensively during the pastdecade as examples of human fibroblasts malignantly transformedby treatment with a chemical carcinogen, 4-nitroquinoline-1-oxide.Recently, in comparing the HuT-1, HuT-12 and HuT-14 cell lineswith KD cells, McCormick and Maher (Mutat. Res., 199, 273–291,1988) found evidence that the malignant cells could not havebeen derived from the latter. But, this did not rule out thepossibility that as the target cells for his original studyof carcinogen-induced transformation, Kakunaga had inadvertentlyused cells from some other, unidentified normal individual.Since the donor of such cells would not be known and the originalcell line was not available, it would be impossible to determinethe degree of identity between such a target cell line and theHuT cell lines. However, in the course of examining methodsfor such testing, we recently became aware that the isozymepattern of these HuT cell lines was identical to that of thehuman fibrosarcoma-derived cell line 8387 established in 1966.We here report that the HuT cell lines and the 8387 cell linealso exhibit an identical series of HLA determinants and identicalrestriction fragment length polymorphisms(RFLPs). Assuming thateach of these three assays measures independently inheritedcharacteristics, the chance that an unrelated donor of the fibroblaststhat gave rise to the HuT cell lines happened to possess characteristicsidentical to those of the patient whose fibrosarcoma gave riseto the 8387 cell line is 1 x 10^–8. Therefore, we concludethat 8387 cells are the source of the malignant cells designatedHuT from Kakunaga's original transformation experiment. AdditionalRFLP analysis, using a probe made from M13 bacteriophage DNAwhich detects a hyperpolymorphic ‘minisatellite’pattern in human DNA, also showed that DNA from HuT-14 cellsand from 8387 cells exhibit identical banding patterns, indicatingthat the cell lines were taken from the same individual.Thelatter banding patterns differed from that observed with DNAfrom KD cells. Karyotyping studies showed that HuT-12 and HuT-14cells have six marker chromosomes and that these are identicalto the six marker chromosomes seen in the 8387 cells. The resultsemphasize the necessity of validating in vitro transformationexperiments by showing that the parental cells and the malignantcells have a common origin.     

Malignant lymphoma antigen expressed in nude mouse tumor cells derived from carcinogen-transformed Bloom's syndrome B-lymphoblastoid cell lines

From nude mouse tumors, in which malignantly transformed Bloom's syndrome (BS) B-lymphoblastoid cell lines were successfully transplanted into s.c. tissues, we have detected strong expression of malignant lymphoma (ML)-associated antigen on the cell surface, by using diluted sera of ML patients and indirect immunofluorescence. Even though carcinogen-treated BS B-lymphoblastoid cell lines expressed various types of cancer antigens (ML, ovarian cancer, stomach cancer, lung cancer, liver cancer, etc.) on the cell membrane as a mixed population (Y. Shiraishi and H. Soma, Proc. Natl. Acad. Sci. USA, 85:8211-8215, 1988), the finding that BS malignant cells originating from nude mouse tumors expressed specific ML-associated antigen seemed significant for ML diagnosis. BS nude mouse tumors were successively transplantable from nude mice to nude mice (100%). Histopathological studies using an electron microscope demonstrated that most tumor cells in s.c. tissues of nude mice were lymphoid malignant cells. Gel electrophoresis and Western blotting analyses demonstrated that the antigen which characterized ML was a single band (Mr 97,000) and did not cross-react with the sera of other cancer patients or with normal sera. Chromosome analysis showed that the cell clones with ML-associated antigen had marker chromosomes involving t(6;?)(p25;?),t(9;?)(q34;?), del(10)(p13),t(12;14)(q24;q11). The expression of ML-associated antigen was also discussed in relation to the marker chromosomes.     

Relation between p53 expression of human colon cancer cell lines and induction of apoptosis by anticancer drugs

The relation between p53 expression of human colon cancer cell lines and induction of apoptosis by anticancer drugs was examined experimentally. HCT116 had wild-type p53 expression and 5-fluorouracil (5-FU) in combination with cisplatinum (CDDP) increased both Sub G1 DNA content and the proportion of apoptotic cells. However, solitary administration of these drugs altered neither. Alternatively, for SW480 with mutant-type p53 expression, both combined administration and solitary administration of anticancer drugs showed minimal effect on Sub G1 DNA content and proportion of apoptotic cells. These results indicate that the type of p53 expression might play an important role in the determination of response to low-dose CDDP + 5-FU therapy in colon cancer patients.     

Regulation of hypoxanthine DNA glycosylase in normal human and Bloom's syndrome fibroblasts

The regulation of the base excision repair enzyme hypoxanthine DNA glycosylase was examined in normal human skin fibroblasts (NHS) and fibroblasts from a patient with Bloom's syndrome. Using randomly proliferating cells and those synchronized at specific intervals in the cell cycle, enzyme levels were shown to become elevated severalfold in a proliferation-associated manner. In NHS synchronized in G0 by serum deprivation or in G1 by isoleucine deprivation, maximal enzyme levels were reached prior to maximal rates of DNA synthesis. In Bloom's syndrome cells synchronized in this manner, these two activities were coincident. Cells synchronized at the G1-S border by hydroxyurea exhibit an initial wave of DNA synthesis upon removal of the drug. The cells then undergo another DNA synthetic cycle climaxing 18-21 h after release. Maximal hypoxanthine glycosylase activity of hydroxyurea-synchronized Bloom's cells was observed during the second round of DNA synthesis. However, in NHS the peak of enzyme activity was observed as early as 9 h prior to the second round of DNA synthesis. To determine if hypoxanthine glycosylase could be induced in the absence of DNA synthesis, serum-synchronized NHS were released in the presence of hydroxyurea. The results showed that inhibition of DNA synthesis did not diminish glycosylase induction which demonstrated that DNA replication was not required for glycosylase induction.     

Transition of phenotypic dimorphism with regard to spontaneous sister chromatid exchange in Epstein-Barr virus-transformed Bloom's syndrome lymphoblastoid cell lines.

We recently established four lymphoblastoid cell lines (LCLs) by infecting the peripheral blood of four Japanese patients suffering from Bloom's syndrome (BS) with Epstein-Barr virus (EBV). During the course of propagating these cell lines, two of them exhibited dimorphism regarding spontaneous sister chromatid exchange (SCE), i.e., a mixed population consisted of cells with extremely high SCE levels characteristic of BS and cells with low SCE levels indistinguishable from that of normal control cells. On the other hand, the other two cell lines maintained a monomorphic population with high SCE levels at least until 30 weeks after EBV infection. The proportion of the cells with high SCE levels in the cell lines with dual phenotype declined as the population doubling numbers (PDN) increased with time and they became ultimately undetectable. The proportion of cells with low SCE levels at the time of EBV infection was estimated in one of these LCLs as 0.075% by extrapolating the linear regression of the logit for the proportion plotted against PDN. In view of the well-known stability of the monomorphic phenotype in representative BS LCLs during extended cultivation, together with the present observations on the dual phenotype, we conclude that the frequent establishment of BS LCLs exclusively with low spontaneous SCE levels is attributable to the various proportions of low-SCE cells existing in vivo in the B-lymphocytes pool of BS individuals and to the selective pressure against the high-SCE cells in in vitro cultures.     

Relation between nucleolar size and growth characteristics in small cell lung cancer cell lines.

Relationships among cytological features, doubling time, S-phase percentage, expression of myc-family oncogenes, DNA ploidy and biochemical properties were studied in thirteen small cell lung cancer cell lines. Six cell lines that grew slowly (average doubling time 99 h) and had lower S-phase percentages (average 32%) showed inconspicuous nucleoli (average area of 1.5 microns 2), and the remaining seven cell lines that grew quickly (average doubling time 45 h) and had higher S-phase percentages (average 44%) showed large and prominent nucleoli (average area of 6.1 microns 2). DNA index value obtained from flow cytometric DNA histograms showed that all cell lines except for H-69 cell line displayed aneuploidy. Ribbon-like cell arrangements were observed in the 7 cell lines that grew quickly, and in 1 cell line that grew slowly. Biochemically, six slow-growing cell lines and four fast-growing cell lines showed high levels of aromatic L-amino acid decarboxylase activity, while in the remaining three fast-growing cell lines its level was low. A high level of c-myc or N-myc oncogene expression was observed in all 7 cell lines that grew quickly, but not in any of the 6 cell lines that grew slowly. It appears that small cell lung cancer cell lines that grow quickly can be expected to have large nucleoli and ribbon-like cell arrangements and to express high levels of myc-family oncogenes, and that nucleolar size is a good indicator for growth characteristics.     

Impact of mitochondrial DNA on hypoxic radiation sensitivity in human fibroblast cells and osteosarcoma cell lines

The purpose of this study was to evaluate the impact of mitochondrial DNA (mtDNA) on radiation sensitivity under hypoxic conditions. The cell lines used were rho+ and rho0, which carry wild-type mtDNA and no mtDNA, respectively. The rho0 cells do not utilize oxygen because they lack the capacity to carry out oxidative phosphorylation. To confirm the role played by mtDNA in different cell lines, two types of cell line were used: human fibroblast and osteosarcoma cells. Radiosensitivity was evaluated by the colony formation assay, micronucleus (MN) formation assay and comet assay. Hypoxia lowered radiosensitivity in all three experiments for all four cell lines. Between rho+ and rho0 cells, no difference was found in the results from the colony formation assay and comet assay. However, higher MN formation was found in rho+ cells than in rho0 cells, not only under room air conditions in both the fibroblast and osteosarcoma cell lines, but also under hypoxic conditions. Therefore, although hypoxia lowers the radiosensitivity in general, the impact of mtDNA persists under hypoxic conditions.     

Enhanced deoxyribonuclease activity in human transformed cells and in Bloom's syndrome cells

Human hereditary diseases such as xeroderma pigmentosum, Fanconi's anemia, ataxia telangiectasia, and Bloom's syndrome are characterized by a proneness for developing cancer associated with abnormalities in the processing of DNA damage. The molecular defects responsible for predisposing human tissues to cancer are still not well understood, despite the fact that a considerable amount of work has already been done on this problem. In this paper, we show that in human tumor cell lines, in cells transformed by DNA tumor viruses, and in cells derived from certain cancer-prone disorders, the level of activity of a 42-kDa deoxyribonuclease is many times higher than in diploid untransformed control cells. This suggests that this activity is linked to, or may play a role in, malignant transformation.     

Discrimination between human melanoma cell lines by fluorescence anisotropy

The fluorescence polarization of dipheylhexatriene (DPH) and trimethylammonium diphenylhexatriene (TMA-DPH) was measured when these markers were imbedded in cells of the human melanoma cell lines IGR37, IGR39, IGR3 and IGRA4, as well as in cells of the mouse melanoma cell lines B16 F1 and B16 F10. These measurements were performed on cell cultures which were grown on quartz plates as well as on cell suspensions. Considerable differences are found between the polarization values of the human cell lines that are related to their different origins. Differences for the plated cells are considerably greater than those for the suspensions. No differences in the polarization values were found for the two mouse melanoma lines. It is concluded that differences in lipid structural order can be found between cell types endowed with different metastasizing capabilities.     

Malignant transformation of an infinite life span human fibroblast cell strain by transfection with v-Ki-ras

To determine if human fibroblasts can be transformed into malignant cells by transfection of a K-ras oncogene, we transfected the provirus of Kirsten murine sarcoma virus (v-Ki-ras) into an infinite life span human cell strain, MSU-1.1, which has a normal morphology, is not anchorage independent, and has a stable, near-diploid karyotype. The transfected populations gave rise to distinct foci composed of morphologically-altered cells. The cells from several independent foci were isolated, propagated, and assayed for anchorage independence and/or tumorigenicity. They formed large-sized colonies in soft agar at a high frequency. Cell strains derived from colonies isolated from agar as well as focus-derived cell strains were injected subcutaneously into athymic mice to test for tumorigenicity. One cell strain yielded myxoid fibromas, the rest produced well-differentiated, progressively-growing, invasive, myxoid or spindle cell sarcomas. The karyotype of each of the cell strains tested, including cell strains derived from tumors, was identical to that of non-transfected MSU-1.1 cells. Two focus-derived strains, and two cell strains derived from sarcomas produced from them, were tested and shown by DNA and RNA hybridization to contain and express the v-Ki-ras oncogene. Radioimmunoprecipitation analysis showed that these strains expressed ras-specific p21 products not found in non-transfected MSU.1.1 cells. When injected intraperitoneally, a cell strain derived from a myxoid tumor gave rise to invasive myxoid tumors at various sites in the body. The same cell strain gave rise to invasive spindle cell sarcomas when injected into the tail vein of the animals.