John W. Littlefield

Elevated dihydrofolate reductase messenger RNA levels in methotrexate-resistant BHK cells

Polysomal RNA from cultured sublines of baby hamster kidney (BHK) cells directed protein synthesis in an in vitro system derived from wheat germ extract. One product of the in vitro synthesis was dihydrofolate reductase (DHFR), as confirmed by methotrexate-substituted Sepharose affinity chromatography followed by SDS-polyacrylamide slab gel electrophoresis and autoradiography of the proteins labeled with 35S-methionine. The DHFR synthesized in vitro comigrates in the gel with authentic BHK DHFR, indicating that the molecular weights and structures of the in vivo and in vitro enzymes are probably the same. Polysomal RNA obtained from the methotrexate-resistant BHK subline (A5), which possesses some 140 times higher DHFR levels than the methotrexate-sensitive parents subline (B1), directed the synthesis of approximately 70 times more DHFR per unit of total in vitro synthesized protein than did B1 polysomal RNA. Assuming then that the rates of translation of A5 and B1 DHFR mRNAs in the wheat germ cell-free system are the same, our results show that a major part of the high DHFR levels observed in A5 cells is due to the presence of elevated quantities of DHFR mRNA.

Mutagen-induced diploid human lymphoblast variants containing altered hypoxanthine guanine phosphoribosyl transferase.

The human lymphoblast line MGL8 was treated with HAT and subsequently “mutagenized” with EMS (200 μg/ml) to give 15% survival, and 6-thioguanine-resistant cells were selected by cloning in soft agarose containing the drug (1 μg/ml). Eighteen sublines of independently derived resistant clones were isolated and studied in detail. One subline had a low residual HGPRT activity of about 1% of the parental cells. The HGPRT of this subline had a higher Km for PRPP, was more sensitive to heat, and was degraded faster by trypsin than the enzyme in extracts of MGL8 cells. This resistant subline and three others contained CRM levels of 1-38%, compared to the wild-type, so they probably represent true structural mutants of the HGPRT gene. All the variants maintained the karyotype of the parental line (46, XY, 6p−).

PARTIAL TRISOMIES IN TWO SPONTANEOUSLY ARISING LONG-LIVED HUMAN KERATINOCYTE LINES

SummaryDuring experiments concerning the introduction of oncogenes into normal human keratinocytes, we observed long-lived colonies arising spontaneously at the same low frequency in control cultures as in those transfected with Ha-rasEJ or activated c-myc or both. Two of these were karyotyped early in their life span and showed additional chromosomal material on the short arm of chromosome 9 in one case and of chromosome 18 in the other, whereas the parental cells had a normal karyotype. This indicates the presence of a partial trisomy in each line, although the origin of the extra chromosomal material is not known. A similarly long-lived human keratinocyte line containing an isochromosome of the long arm of chromosome 8 has been described elsewhere. Together these results suggest that the spontaneous occurrence of long-lived lines is more common in human keratinocytes than in fibroblasts and that a triple dose of one or more genes may be the initial event in this process.

Rescue of terminally differentiating teratocarcinoma cells by fusion to undifferentiated parental cells.

A new clonal CBA mouse teratocarcinoma cell line called H6 grows rapidly without calcium in suspension culture. When attached to a gelatin-coated surface in the presence of calcium, differentiation to large flat cells, which secrete plasminogen activator, occurs after exposure to retinoic acid. Differentiation is terminal after about six cell divisions. H6 variants resistant to 6-thioguanine, 5-bromodeoxyuridine, ouabain, chloramphenicol, retinoic acid, or combinations thereof, have been isolated. Cell hybridizations were made between undifferentiated stem cells and the differentiated derivatives of H6. These experiments indicate that the undifferentiated cells can “rescue” cells in the initial stages of differentiation and that the signs of differentiation are soon lost in such hybrids. Hybridization without “rescue” may also occur, as suggested by small abortive colonies of large cells.

Calcium-induced compaction and its inhibition in embryonal carcinoma cell aggregates☆

H6 embryonal carcinoma cells form aggregates of cells in culture medium which contains 2 mM calcium. These aggregates are described as uncompacted, indicating that the individual cells of the aggregate are spherical and are in limited contact with each other. In contrast, compaction of the aggregate, induced by increasing the calcium concentration, results in a tight mass of cells flattened against one another and connected by intercellular junctions. At least 85-97% of the aggregates undergo compaction in 7 mM calcium and are subsequently decompacted if removed to 2 mM calcium. Since calcium ionophore A23187 does not induce compaction, extracellular rather than intracellular calcium seems to be the limiting factor. We have demonstrated that this calcium-induced morphogenetic change is sensitive to inhibition by agents which also prevent the calcium-dependent compaction of the 8-cell mouse embryo. The cytoskeletal-binding drugs tetracaine HCl, colcemid, vinblastine, colchicine, and cytochalasin B each inhibit compaction of H6 aggregates. Interference at surface molecule sites by exposure to the lectins wheat germ agglutinin or concanavalin A or by interruption of glycosylation with exposure to tunicamycin, or by reaction with anti-H6 Fab or anti-F9, also prevent compaction. Since the mouse embryo and embryonal carcinoma cells share certain processes which are involved in initiating and maintaining compaction, these processes and their subsequent roles in differentiation may be examined using embryonal carcinoma cell aggregates.

Endoderm-secreted factor stimulates growth of embryonal carcinoma stem cells

SummaryStem cells of the embryonal carcinoma cell line called H6 can be induced to differnetiate to endoderm-like cells by retinoic acid (3×10−6M). We have detected a diffusible and stable factor which is secreted by H6 endoderm-like cells and stimulates the growth of H6 stem cells. The stimulation by the endoderm-like cells is considereably greater than that by mouse fibroblasts or H6 stem cells themselves. No reciprocal stimulation of endoderm-like cells by stem cells occurs. Part but not all of the stimulation might be due to extracellular matrix proteins or to insulin-like growth factor type 2, each of which also stimulates the growth of H6 stem cells. Insulin causes no such stimulation.

Origin of chromosome rearrangements in two long-lived human keratinocyte lines

SummaryWe have determined the origin of the extra chromosomal material in the karyotypes of two spontaneously-occurring long-lived human keratinocyte lines, HKC-N2 and HKC-N6. In each case the extra material was derived from the chromosome on which it was located. Possible relationships between the triplication of chromosomal material and the overcoming of cell senescence are discussed.

RNA from rat hepatoma cells can activate phenylalanine hydroxylase gene of mouse erythroleukemia cells.

Mouse erythroleukemia (MEL) cells do not synthesize any detectable level of phenylalanine hydroxylase and thus do not grow in Tyr− medium. Rat hepatoma cells that constitutively express phenylalanine hydroxylase were treated prior to fusion with MEL cells with biochemical inhibitors to inactivate different macromolecular components of the cells, and the fusion products were selected in Tyr− medium. Continuously growing populations of cells resembling the parental MEL cells and expressing mouse phenylalanine hydroxylase were obtained only when rat hepatoma cells treated with mitomycin or iodoacetamide, which inactivate DNA and SH proteins, respectively, were fused with MEL cells. Fusion of MEL cells with UV-treated rat hepatoma cells did not result in the activation of the mouse phenylalanine hydroxylase gene. UV treatment damages both DNA and RNA. These data suggested that RNA was involved in the regulation of phenylalanine hydroxylase gene. Additional evidence for the role of RNA in the phenylalanine hydroxylase gene regulation was obtained from RNA transfection studies. RNA only from cells which express phenylalanine hydroxylase, such as rat hepatoma cells and MEL cybrids, when introduced into MEL cells by the CaPO4 coprecipitation method, resulted in the permanent activation of the mouse phenylalanine hydroxylase gene.

Absence of uvomorulin in a slowly compacting variant of H6 embryonal carcinoma cells

When allowed to aggregate in calcium-containing medium, the H6 embryonal carcinoma cell variant named 6B(NG)C25 compacted more slowly than wild-type cells, and aggregates of hybrids between it and wild-type cells also compacted slowly, as if the variation (mutation) acted in a dominant fashion. In agreement with this, we now have found that the cell adhesion molecule uvomorulin is markedly reduced or absent in 6B(NG)C25 cells, as well as in the hybrids. A small amount of a higher-molecular-weight protein reacting with the antibody is present, which might represent residual uvomorulin migrating at a slower rate, an altered uvomorulin, the known precursor to uvomorulin, or an unrelated cross-reacting protein.

Analysis of HGPRT − CRM + human lymphoblast mutants

Three 6-thioguanine-resistant mutants of the human diploid lymphoblast line MGL-8 were studied. The inactivation by heat of both HGPRT activity and antigenicity of the HGPRT immunologically cross-reacting material of the A30 mutant cells were not protected by PRPP, indicating that the HGPRT in A30 cells has an altered PRPP binding site, leading to lack of stabilization and rapid degradation of the enzyme. Two dimensional separations of the immunoprecipitates from extracts of the parental and mutant cell lines showed that the A35 mutant CRM has a more acidic isoelectric pH, while the A30 CRM has a more basic isoelectric pH and that the A30 protein has a faster rate of degradation than the wild-type HGPRT. The A30 CRM also has a smaller molecular size than the wild-type enzyme.