Jana Hillova

Infectivity of Rous Sarcoma Cell DNA: Comparison of Two Techniques of Transfection Assay

A DNA extracted from a clone of chicken cells transformed by the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup D(SR-RSV-D), was assayed for infectivity by means of DEAE-dextran and calcium techniques. The calcium technique like the previously described DEAE-dextran procedure gave rise to viruses in transfection assays with both native and denatured (S1 nuclease susceptible) DNAs. The efficiency of these transfection techniques with native DNA was compared and found to be about the same provided that with the calcium technique carrier DNA was used to complement DNA concentrations lower than 2.5 mug/ml.

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.

Transformation-defective mutants with 5' deletions of the src gene are frequently generated during replication of rous sarcoma virus in established quail fibroblasts

Replication of Rous sarcoma virus (RSV) in avian fibroblasts leads to the generation of replication-competent variants that are defective for cell transformation (td virus). These td variants contain deletions affecting various portions of the v-src gene. We compared the rate of td virus production in Q3B cells, a quail cell line established by mutagen treatment, and in normal quail fibroblasts. Twenty-five days after infection with an RSV stock containing only transforming virions, Q3B cells harbor similar amounts of v-src-containing and v-src-deleted proviruses. However, these cells synthesize very low levels of p60v-src and generate large excess of td variants, as determined by biological assays. Unlike Q3B cells, normal quail fibroblasts infected with the same virus stock produce td variants only after multiple passages of undiluted virus on fresh cells. Restriction analysis showed that the td virus produced by Q3B cells is composed of two types of genomes: one lacking the entire v-src gene and the other carrying partial deletions of this gene predominantly located in the amino-terminal portion of the coding region of v-src. To study the mechanisms of these partial deletions, we molecularly cloned and sequenced the v-src genes of several td proviruses. We show that these mutants carry single or multiple v-src deletions of limited size, presumably generated by multiple mechanisms. Two deletions of 170 and 112 bp located in the 5 portion of v-src are frequently generated during RSV replication in Q3B cells and may represent preferential sites for v-src deletion in these cells.

Regression of the malignant aspect of intraepithelial neoplasias following an LH—RH agonist treatment and detection of human papillomavirus by molecular hybridization

Patients presenting genital intraepithelial neoplasia and/or flat condyloma were treated with DTrp6-LH-RH (triptorelin) to induce a transitory suppression of estrogens. This treatment led in some cases to a complete clinical and histological regression accompanied by a disappearance of human papillomavirus sequences as detected by molecular hybridization.

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.

Presence of human papilloma virus types 16 and 18 in genital warts and cervical neoplasias

Certain types of human papilloma viruses (HPV) are associated with human genital proliferative diseases, and among them HPV16 and HPV18 seem to play an important role in the occurrence of cervical cancer. We used restriction enzyme analysis and molecular hybridization, in order to investigate the type of viral infection and the physical state of viral DNA in gynecological benign, pre-malignant and malignant lesions. HPV6/11 specific sequences could only be detected as episomes and this in benign lesions classified as condylomata acuminata. On the other hand, HPV16 and HPV18 sequences were detected in non-malignant lesions such as flat condylomata (7 out of 14 cases), pre-malignant lesions including cervical intra-epithelial neoplasias (10 out of 20 cases), and most frequently in cervical invasive cancers (21 out of 27 cases). In a large number of virus-positive cases, HPV16 and HPV18 could only be discerned in forms consistent with the existence of episomes and/or randomly integrated head-to-tail oligomers. However, some invasive carcinomas and cervical intra-epithelial neoplasias contained, in addition, clonal outgrowths with detectable virus-cellular junction fragments of the integrated viral genomes. In the light of these data, monitoring the type of viral infection proves to be an important adjunct to histological analysis when assessing those patients affected by condyloma or cervical intra-epithelial neoplasia who are at risk for developing invasive carcinoma.

RSV Provirus with Same Flanking Sequences Is Found on Different Size Classes of Chinese Hamster Chromosomes

Rous sarcoma virus(RSV)-transformed Chinese hamster fibroblasts, containing approximately ten copies of the DNA domain comprising a single provirus and its flanking cellular sequences, were arrested in metaphase, and the chromosomes were fractionated by size in a sucrose gradient. The resolution of polymorphic ribosomal genes, the dihydrofolate reductase gene, and the c-src gene demonstrated that the gradient can distinguish between small, medium, and large chromosomes. The same DNA domain carrying the RSV provirus was found to be associated with chromosomes of all three size classes. Polymorphic copies of the domain in small and large chromosomes could be distinguished from those in medium-sized chromosomes because of the polymorphism in the XhoI and EcoRI sites on 5 and 3 adjacent cellular sequences, respectively. The presence of the same provirus domain on different chromosomes, together with the karyological data showing trisomies in the same chromosome size classes, suggest that the provirus domain, possibly the entire replicon, was duplicated and transferred to different nonhomologous chromosomes, and the transfer was followed by duplication of the target chromosomes. The possibility that proviral long terminal repeats might be involved in replicon transfer is discussed.

Two-Hit Kinetics of Focus Formation in Cells Transfected with Rous Sarcoma Provirus

DNA was purified from an established line of SR-E-transformed quail cells and was assayed for transfection in turkey cells. Transfection events were determined from foci of transformed cells growth under agar overlay which was used to prevent the spread of the progeny virus. It was found that transfection with Rous sarcoma proviruses follows two-hit kinetics, indicating that at least two (unlinked) provirus are required to give rise to a focus of transformed cells.

Isolation of a line of immortal chicken embryo fibroblasts after transfection with the nuclei of Rous sarcoma virus-transformed Chinese hamster cells☆

Secondary cultures of chicken embryo fibroblasts were transfected with purified nuclei from lysed cells of a clonal line of temperature-sensitive Rous sarcoma virus (tsRSV)-transformed Chinese hamster fibroblasts. After propagation for 3 months an established cell line designated ChR32 was obtained in one chicken cell culture. The cells of this line have been propagated so far for 18 months, whereas normal chicken embryo fibroblasts died after 2 months. The established cells were heteroploid with a diploid modal number of macrochromosomes and two Z chromosomes. No Chinese hamster chromosomes could be identified. Southern blot analysis of DNA from the uncloned ChR32 cells and the clones provided evidence that these established cells were, in fact, clonal in origin and contained full-length RSV proviruses and no defective proviruses. Furthermore, they contained, at the 3 end proviral-cellular junction, Bg/II, HpaI, KpnI, SacI, and XbaI fragments of the same size as the Chinese hamster donor cells, suggesting that the cellular sequence adjacent to the provirus is of Chinese hamster origin. The cells after establishment were able to grow continuously at 37 degrees or 41 degrees C and produce a large amount of ts sarcoma virus particles. A corollary finding was that these virus particles were non-leaky for the transforming function at the non-permissive temperature.

Tandem integration of avian retroviruses in double-infected cells.

Chicken embryo fibroblasts were chronically infected by both RAV-1 and SR-E, and established SR-E-transformed quail cells were superinfected with RAV-1. About 75 and 50% of double-infected and superinfected cells, respectively, produced both parental viruses. The DNA purified from these cells contained infectious provirus of both parents and no detectable recombinant provirus. In assays using agar overlay, about 5% of the transformed foci that were induced by the DNA of double-infected, but not superinfected, cells were found to contain the progeny of both parents. This observation suggests that in double infections with avian retroviruses, DNA of both parent viruses may integrate into the host genome either in tandem or in close proximity.