Jørgen Fogh

Human-tumor-derived cell lines contain common and different transforming genes

We have screened different cultured cell lines established from human tumors for the ability of their DNAs to induce transformed foci in NIH/3T3 cells. Based on restriction endonuclease digestions and the presence of human sequences in mouse transformants, we conclude that five of these human tumor cell lines contain a gene or genes capable of transforming mouse cells and that at least three different transforming genes are present in these five lines. Three cell lines, two derived from lung carcinomas and one derived from a colon carcinoma, transfer the same or closely related human genes. If these transforming genes are mediating the tumorigenic state of the human cells, then our results indicate that overlapping pathways leading to tumorigenesis may arise independently.

New human transforming genes detected by a tumorigenicity assay.

We have developed a sensitive bioassay for transforming genes based on the tumorigenicity of cotransfected NIH3T3 cells in nude mice. The assay differs substantially from the NIH3T3 focus assay. Using it, we have detected the transfer of three transforming genes from the DNA of MCF-7, a human mammary carcinoma cell line. One of these is N-ras, which is amplified in MCF-7 DNA. The other two, which we have called mcf2 and mcf3, do not appear to be related to known oncogenes. We cannot detect their transfer by using the NIH3T3 focus assay. We do not yet know whether either mcf2 or mcf3 is associated with genetic abnormalities in MCF-7 cells.

The Nude Mouse in Cancer Research

Publisher Summary The nude mouse is considered a marvel as a laboratory animal and in many respects is uniquely qualified for in vivo model studies of human cancer. Most human tumors have been transplanted to nude mice and tumor lines are established with varying degrees of ease. It is noted that the additional immunosuppression of the mice or the use of very young animals can increase tumor growth and malignant expression. Other variables that affect tumor transplantation include the health of the mice, site of injection of the transplant, and specific properties or requirements of the human tumor cells. Hormone-dependent human tumors are grown in nude mice, either in special sites or after hormonal supplement. It is observed that the tumor growth rates differ in nude mice and in cancer patient and this tumor heterogeneity occasionally result in the growth of selected tumor components. These differences do not necessarily demean the human tumor-nude mouse model, but is well in line with the changes occurring with time in the human patient tumors. Many tumor characteristics appear to be stable in the nude mouse, and it has been shown that tumor markers and secretion of certain hormones may be maintained in the xenografts.

A REVIEW OF CELL CULTURE CONTAMINATIONS

It is apparent that most laboratories employing tissue and cell cultures for experimental or diagnostic purposes encounter contaminations. If not detected and properly controlled or eliminated, contaminants may severely affect such investigations, and be the cause of incorrect interpretations. The Committee on Contaminations in Cells and Tissue Cultures of the Tissue Culture Association considered it as one of its functions to review the most pertinent literature related to contaminations with cells, bacteria, yeasts, molds, parasites, Mycoplasma, and viruses. The present review article is not intended to cover the subject completely but will, hopefully, be helpful to workers in the area of cell and tissue cultures who are concerned about the problem of contamination. Apparently many are not thoroughly familiar with the proper procedures for prevention, detection, and, perhaps, elimination of contaminants from the experimental systems that they employ.

Nonrandom chromosome alterations in rhabdomyosarcoma

Chromosome banding analysis was attempted on tumor cells from a total of six rhabdomyosarcomas. Results revealed a variety of chromosome alterations, including frequent structural rearrangement of chromosome #1 and the finding in one patient of multiple double minutes. The single chromosome most consistently involved in structural rearrangements was #3. Simple deletion or translocation of either the long or short arm of chromosome #3 was found in all rhabdomyosarcoma tumors examined in this report. Further, a review of the limited previous literature on rhabdomyosarcoma provided further support for the frequent alteration of chromosome #3 in this disease. Results from our study provide preliminary evidence that alterations of chromosome 3p14-21 may represent a site of nonrandom chromosome change in rhabdomyosarcoma.

Human Tumor Lines for Cancer Research

(1986). Human Tumor Lines for Cancer Research. Cancer Investigation: Vol. 4, No. 2, pp. 157-184.

A comparison of methods for morphological studies of cultured cells

SummaryA number of fixation methods for different types of cells in culture were compared, and the best preservation of nuclear and cytoplasmic details was obtained by fixation with Bouins solution for 15 min, prior to staining with hematoxylin and eosin. All of the fixatives, including Bouins solution, damaged various structures, notably the peripheral glas-attached cytoplasm and the intercellular connections. Micrographs obtained by bright field, phase contrast, and interference contrast (Nomarski) microscopy are presented. Much more realistic pictures, bringing out details not observed after fixation and staining, were obtained by Nomarski microscopy of living, unfixed cultures. Most conspicuous were numerous thin, cytoplasmic, cilia-like extensions, concentrated on the glass-attached peripheral margins, which were also visible on other cell surfaces and as intercellular connections. These structures were most characteristic of SV40-transformed human amnion cells. Although fixation and staining emphasize certain cell components (for example, inclusion bodies), many aspects of cellular morphology are better demonstrated by observing living cells by interference microscopy or by Nomarski interference contrast microscopy. Surface features of unfixed cells, seen by Nomarski interference contrast microscopy, were similar to the surface features of glutaraldehyde-osmium tetroxide-fixed cells studied as metallic replicas in the electron microscope.

Growth in Vitro of Mycoplasma-Infected Human Amnion Cells, FL Amnion Cells, and Mycoplasma-Modified FL Cells

Summary The population doubling time for lines of FL human amnion cells infected with mycoplasma fermentans (strain HT) was increased (30 hr) compared to uninfected FL cells (17.5 hr). Infected FL cell lines from which mycoplasma was eliminated, had a doubling time of 21 hr and showed an increased resistance to reinfection, even several years after the mycoplasma elimination. Although the amounts of cell-associated mycoplasma were similar to those of infected FL cells, cytopathic effects and cell destruction were much less pronounced after reinfection of such lines. The amounts of cell-associated, as well as free mycoplasma, were reduced in cultures of primary amnion cells as compared to the transformed cells. Primary amnion cells were highly resistant to mycoplasma infection as judged by the number of glass-attached cells and lack of cell destruction. The theoretical and practical implications of these observations are discussed.

Mycoplasma effects on SV40 transformation of human amnion cells.

Summary Incidence of transformation of human amnion cells by SV40 was reduced and the appearance of transformed foci delayed by concurrent SV40-mycoplasma infection of primary cultures. The number of population doublings during serial cultivation was reduced, as was the number of culture passages and the time before “crisis” of the SV40 transformed cells. Mycoplasma infection of an SV40 transformed strain already in serial cultivation caused even more cell destruction and effects on the population doubling number. The virus production pattern changed, but differently for individual strains. After mycoplasma elimination, the number of passages and cell divisions, and the time before “crisis” increased, and several strains were recovered from “crisis” as permanent cell lines.

Chromosomes of SV40 transformed human amnion cells after mycoplasma infection.

Summary Concurrent mycoplasma-SV40 infection of cultures of primary amnion cells changed the chromosome picture of the resulting SV40 transformed cells. Chromosome numbers were maintained in the diploid range for a longer period, and most abnormalities were present at lower frequencies, especially during the later period of cultivation prior to “crisis.” Chromosome numbers and frequencies of abnormalities in the mycoplasma-modified cells were not reversed after mycoplasma elimination. However, a large telocentric chromosome was more frequent after mycoplasma elimination. The recovery of several post-“crisis” cell lines from a mycoplasma-modified SV40 transformed strain of cells may be correlated with the lower chromosome numbers and the fewer abnormalities.