Régine Mariage-Samson

Amplification of the provirus region in Rous sarcoma virus-transformed Chinese hamster cells and segregation of the amplified copies in somatic cell hybrids

Four independent clones of RSV-transformed Chinese hamster fibroblasts were isolated. Southern blots and dot hybridization studies showed that in three out of four clones there were four to eight times as many integrated proviruses as in the fourth clone which contained at least one complete provirus. Restriction mapping studies showed that although the integration site varied from clone to clone, all the proviral copies in the same clone shared the same flanking cellular sequences. In one clone there are at least two polymorphic proviral variants A and B, one with and one without a BglI site. Further experiments were performed to see if the variants could be physically separated. RSV-Transformed Chinese hamster cells resistant to thioguanine were fused with mouse cells to give somatic hybrids which preferentially segregate Chinese hamster chromosomes. Ten out of eleven hybrids positive for virus rescue have lost up to 90% of the parental provirus copies. Four of these hybrids were found to contain the provirus variant A alone, one the variant B alone, and the rest contained both variants. All the proviruses retained in somatic hybrids shared the flanking cellular sequences of the parental provirus. Provirus segregation in somatic hybrids confirms that multiple (about ten) copies of the provirus region are present in the karyotype of parental RSV-transformed cells and, furthermore, suggests that the amplified copies of this region are translocated to different chromosomes.

Ability of mammalian fibroblasts to grow in synthetic medium containing neither serum nor exogenously added macromolecules

SummaryRous sarcoma virus transformed Chinese hamster fibroblasts, clone CHR1-3, were established at high temperature, then subcloned. Six subclones with round and flat morphology harboring undeleted and partially deleted RSV proviruses, respectively, were seeded into serum-free synthetic medium with no macromolecular additives, and maintained for 2 mo. One flat subclone no. 14, fully designatedsfCHR1-3.14 for itsserum-free phenotype, was further propagated in the same medium. The cells grew exponentially in loosely attached monolayers and dould be serially passaged on bare polystyrene with an average population doubling time of 46 h. Cell attachment could be improved by using collagen-coated polystyrene or by adding a methionine supplement to the culture medium. Furthermore, thesfCHR1-3.14 cells could be subcloned and further grown in nonselective medium. The reversion rate of thesf phenotype was estimated to be 1 to 2%/cell generation. Evidence for an autocrinal stimulation was obtained by cloning efficiency assays showing a requirement for a threshold cell density. Slight growth stimulation could also be detected in assays using conditioned medium fromsfCHR1-3.14 cells and serum-restrictedwild-type (wt)NIH3T3, but notwtCHR1-3.14, cells as indicator cells. Finally,wtNIH3T3 cells used in these assays were assayed for serum-free growth and found to be able to develop their ownsf phenotype; in this respect they resemble the previously establishedsfCHR1-3.14 cells.

Autocrine factor-independent growth of mammalian fibroblasts established in fully synthetic medium: no v-onc requirement in establishment

SummaryIn a previous study Chinese hamster fibroblasts carrying a partially deleted v-src were established in a synthetic medium lacking macromolecular supplements and shown to possess a particular serum-free phenotype hereafter designatedsf. In cloning efficiency assays,sf, unlike wild-type, fibroblasts required a threshold cell density to growth from single cells, suggesting autocrine stimulation. In the present study a conditioned medium harvested fromsf cells was added to the samesf cells, and the resulting cloning density was found to markedly diminish rather than increase.Sf cells were found to be unable to grow at cloning density because of trypsin damage;sf cells seeded into trypsin inhibitor-containing medium cloned with no requirement for threshold cells and were therefore independent of autocrine secretion from neighboring cells. Their cloning efficiency reached 7.7%; this value could not be improved by subcloning thesf culture, and it diminished when selenium was not added to the assay medium. To determine whether v-src is involved in thesf phenotype, five clones of the parental Chinese hamster fibroblast line not infected with Rous sarcoma virus were explanted into serum-free cultures with no macromolecular additives as in the case of v-src-containing cells. Each clone gave rise to ansf cell line growing indefinitely in synthetic medium like the v-src-containingsf cells, showing that the v-src gene is not required either for the establishment or maintenance of thesf phenotype.

Indefinite growth of mammalian cell autotrophs on amino acids, vitamins, and glucose.

Established mammalian cells segregate variants, named autotrophs, able to proliferate in the absence of hormones and proteins of any kind. We provided complete formulas for autotrophic media composed of amino acids, vitamins, glucose, and inorganic salts only and showed that such media are sufficient to sustain continuous propagation of Chinese hamster fibroblasts and human keratinocytes. The cells grew on bare polystyrene and divided every 32 to 44 hours reaching approximately 8 x 10(5) cells/cm2. The results showed that the autotrophs may replace conventional cultures in most experimental and industrial cell production systems.