The action of Mendelian genes in human diploid cell strains

Some of the cells of every human being will grow outside the body as microorganisms. It is possible to show, in a variety of ways, that these cells resemble genetically the individual from whom they were obtained. Over 35 inherited human diseases and anomalies can now be studied in such cell lines. Human diploid cell strains, biochemically marked by one or more mutant Mendelian genes, have proven particularly useful for the study of gene action in man and for the detection of genetic changes such as mutation and somatic cell hybridization. In addition, the strains have a number of clinical applications, including the antenatal diagnosis of inherited disease. The failure of cultured human cells to display their phenotype at most loci continues to restrict their use in both genetics and medicine. There are reasons for hoping that this difficulty will eventually be solved, and some experiments bearing on the problem are already feasible.     

Regulation of IgA secretion by T cell clones derived from the human gastrointestinal tract

The role of T cells in Ig isotype regulation is still unclear. To address this question, we generated mitogen-stimulated T cell clones from normal human lymphoid follicles of the gut-associated lymphoid tissue (appendix). Both the T cell clones and clonal supernatants provided preferential help for IgA secretion by PWM-stimulated B cells. Many of these CD3+, CD4+, 4B4+, DR+ helper clones co-expressed Fc-gamma and Fc-alpha R, but there was poor correlation between the expression of Fc-alpha R and IgA help (p = 0.31). Most of the T cell clones helped both IgM+A- and IgM-A+ B cell populations to secrete IgA, suggesting that they mediate switch of isotype-uncommitted B cells as well as post-switch expansion of IgA-committed B cells; however, some of the T cell clones helped IgM+A- B cell populations much more than IgM-A+ B cell populations, suggesting that, in this case, the regulatory effect is predominantly at the level of B cell switch. In all, these results show that the mucosal immune system contains individual T cells which are capable of positively regulating IgA-specific isotype differentiation at two levels of B cell development, thus allowing for efficient generation of IgA-secreting B cells.     

Interallelic complementation in hydrid cells derived from Chinese hamster diploid clones deficient in hypoxanthine-guanine phosphoribosyl-transferase activity

Activity of hypoxanthine-guanine phosphoribosyl-transferase (HGPRT) has been demonstrated in the hybrid cells formed from the fusion of clonal cells from four Chinese hamster diploid clones, Cl. 250, 252, 253, and 254 cells derived from clonal wild type cells, deficient in this enzyme activity, by the stepwise treatment with 8-azaguanine and 6-thioguanine. The HGPRT-positive cells, characterized by tetraploid karyology, were isolated clonally by the chemical selection (HAT) in 3 hybrid mixtures; Cl. 250 × 254, 252 × 254, and 253 × 254 cells, while none was isolated in another seven hybrid mixtures including 4 homologous mixtures. The enzyme activity was proved by enzymological as well as autoradiographical studies with the use of ³H-hypoxanthine. These results indicate that mutually complementable alterations rather than gene deletions were induced in the 2 cistrons of the locus responsible for the relevant enzyme activity in hamster cells by analog treatment. Interallelic complementation occurred in these hybrid cells.     

Complementation in heterokaryons derived from a thymidine kinase deficient cell line and senescent human diploid cells.

Incorporation of [3H]thymidine was observed in both parental nuclei in heterokaryons resulting from the fusion of post-mitotic, "senescent" human diploid cells and a thymidine kinase-deficient murine cell line (3T3der-4E). The senescent nuclei displayed a sudden increase of activity approximately 4--6 hours after fusion. A moderate increase of thymidine incorporation was observed in 3T3der-4E cells cocultivated with but not fused to senescent cells, consistent with metabolic cooperation. Chromosome preparations of cultures fixed approximately 24 hr after fusion revealed the presence of hybrid metaphase cells containing almost the entire human complement. All of the identifiable human chromosomes were bi-armed, suggesting that the senescent nuclei were stimulated to reinitiate replicative DNA synthesis rather than repair synthesis in these heterokaryons.     

Cell cycles in human diploid and aneuploid strains

Summary Autoradiographic investigation of the cell cycle of 12 diploid and 12 abnormal human fibroblast strains was carried out. Two cell strains (trisomy 7 and monosomy 21) derived from spontaneous abortuses showed prolongation of the G₂ period accompanied by the shortening of the S period. Other cytogenetically abnormal embryonic strains (trisomy 9, 14 and triploidy) did not deviate from the diploid pattern. Three cell strains (LHC-1-70, LHC-6-70, LHC-411) derived from the patients with karyotypes 47,XXX, 47,XY,+18 and 46,XX,5p—respectivly had embryonic types of proliferation with a short G₂ period. In two other strains from the patients with Down's syndrom the G₂ period was prolonged. There was no statistically significant difference in the parameters of the cell cycle between the control and the strains derived from a patient with Klinefelter syndrom and from a male patient with karyotype 46,XX. The modificatory effect of the chromosomal abnormalities on the parameters of cell cycle is discussed.

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Zusammenfassung Der Zellcyclus von 12 normal diploiden und 12 abnormen menschlichen Fibroblasten-Zellinien wurde untersucht. Zwei Zellinien (Trisomie 7 und Monosomie 21) zeigten eine Verlängerung der G₂-Phase und eine Verkürzung der S-Phase. Zellinien (Trisomie 9, 14 und Triploidie) wichen nicht von der Norm ab. Drei Zellinien (47,XXX; 47,XY,+18; 46,XX,5p-) zeigten eine Verkürzung von G₂. G₂ war dagegen verlängert bei zwei Zellinien von Down-Syndrom. Keine Abweichung fand sich bei einem Klinefelter-Patienten und einem männlichen Probanden mit 46,XX.

     

Survival of 60Co-irradiated herpes simplex virus in 15 human diploid fibroblast cell strains

The present study was designed to determine the extent to which herpes simplex virus (HSV) may be utilized to study the repair of DNA damaged by ionizing radiation. We investigated the survival of 60Co-irradiated HSV in cell strains derived from 2 normal controls and 13 patients with a broad range of diseases associated with possible DNA repair deficiencies. Irradiation was performed under two conditions to vary the type of damage incurred by the virus. HSV survival was greatly enhanced when the virus was irradiated in such a way that the indirect effects of ionizing radiation were minimized. We found no correlation between cellular hypersensitivity to ionizing radiation and survival of irradiated HSV. Reduced levels of virus survival were found in only 1 cell strain. When cells were treated with ionizing radiation or UV light prior to infection, no enhancement of virus survival was observed.     

The influence of progressive growth on the specific catalase activity of human diploid cell strains

It was shown previously that the specific catalase activity of human diploid cell strains falls immediately after subculture and then progressively rises in an exponential fashion. In this paper evidence is presented suggesting that the rise in catalase activity cannot be due to an accumulation within the cell of a small molecule which enhances enzyme activity in cell-free extracts. It is also shown that activity per cell, as well as per unit cell protein, rises as the culture grows. The rate of fall of specific catalase activity immediately after subculture is greater if the cells are at a low population density than if they are at a high one. The rate of fall can be made more sharp by increasing the frequency with which the cultures are fed. It is shown that used medium, which has previously been incubated with cultured cells of the same strain, does not significantly change either the rate of fall of specific catalase activity following subculture, or the rate of its subsequent rise. It is postulated, as one possibility, that the cells liberate into the medium an enhancer of cell catalase activity which is highly labile. The steady state concentration of this enhancer in the medium might be expected to increase as the culture grew, but to decrease when the cells are subcultured into fresh medium or when the frequency of feedings is increased.     

Sensitivity of human diploid fibroblast cell strains from various genetic disorders to acute and protracted radiation exposure

The cytotoxic effect of acute X irradiation was studied by a colony formation assay in 114 human skin fibroblast cell strains from 31 apparently normal individuals and 83 patients with a variety of genetic disorders possibly associated with in vitro hypersensitivity to ionizing radiation. The effect of protracted exposure to beta radiation from tritiated water (HTO) was examined in parallel experiments in 65 of these strains. The disorders included neurological diseases and syndromes characterized by an increased susceptibility to spontaneous and radiation-induced cancer. Homozygous ataxia telangiectasia and Nijmegen break syndrome cells were highly sensitive to both types of radiation. However, the response of cells from the other genetic disorders fell within the broad range characteristic of normal cell strains. While HTO may be useful as a quantitative method for determining the cytotoxic response of human diploid cells to ionizing radiation, the present results indicate that it does not offer a more sensitive assay than acute X irradiation for detecting minor degrees of hypersensitivity.     

Isolation and characterization of diploid clones from adult and newborn rat liver cell lines

Summary A high frequency of diploid and near-diploid clones were developed from cell lines derived from adult and newborn rat liver using micropipettes. There were some differences in morphology, biochemical properties and growth rate between clones. Cloned cells had low levels of tyrosine transaminase activity, glucose-6-phosphatase activity and albumin content. A diploid clone and a pseudodiploid clone derived from adult rat liver cell line were positive for α-fetoprotein. This work was supported by a grant for cancer research from the Japanese Ministry of Education.     

Possibilities of vaccine manufacture in human diploid cell strains with a serum replacement factor.

Cell lines MDCK (canine kidney), BGM (Buffalo green monkey kidney) and human embryonic lung fibroblast will support viral growth efficiently in medium without serum. Both MRC-5 and WI-38 cell strains have been approved by the Food and Drug Administration for manufacturing viral vaccines against cytomegalovirus and varicella-zoster virus. In this study we examine these two cell lines and viruses for their ability to grow in medium containing a serum replacement. The serum substitute used is LPSR-1 (low protein serum replacement). Using LPSR-1 in defined medium, we demonstrate multipassage cell growth and viral cultivation and replication equivalent to those obtained in medium containing fetal bovine serum (FBS). Viral growth after complete elimination of FBS varies and depends on cell line and virus. Serum substitutes can eliminate FBS in the viral growth phase of vaccine production and reduce costs.