Periodic fluctuations in proliferation of SV-40 transformed human skin fibroblast lines with prolonged lifespan

A human fibroblastic cell line transformed by the SV40-T antigen sequence and continuously cultured for 7 months displayed large periodic variations in cell proliferation. This contrasted with other characteristics of this cell line that remained constant: mosaic cell shape, absence of cell contact inhibition, and predominance of a hypodiploid population. Similar fluctuations in proliferative capacity were also found during the long-term growth of a transformed but nonimmortalized human fibroblastic line prior to senescence, and in the established hamster fibroblastic Nil cell line. This growth pattern suggests a recurrent stimulation of growth in these three transformed cell lines. The proliferation pattern from cultured transformed cells may thus be complex and requires further investigation. These variations presumably influence major cell functions. This observation has important implications for the analysis of data from such cell lines.Abbreviations I-SF immortalized human skin fibroblasts - T-SF transformed human skin fibroblasts - FBS fetal bovine serum     

Effects of feeder layers made of human,mouse, hamster,and rat cells on the cloning efficiency of transformed human cells

Summary The effect of feeder layers on cloning efficiency of transformed human cells was investigated. Embryonic human skin or lung fibroblasts; adult human skin fibroblasts; early passage cells from embryos of mouse, rat, and hamster; established mouse cell lines; 3T3 and 10T1/2 were used as feeder layers after they were lethally exposed to Co-60 gamma-rays at 3,000 rad. As test cells to study the effect of feeder layers on cloning efficiency, WI-38 CT-1 cells transformed in vitro by Co-60 gamma-rays and HGC cells cultured from a human gastric cancer were used. The effect of feeder layers on the cloning efficiency of the test cells was dependent on cell density of feeder layer cells, sources of the feeder layer cells, and kinds of test cells. An optimal density of feeder cels produced cloning efficiencies 3 to 15 times higher than in cultures without a feeder layer. Generally, high density of cells in feeder layers decreased the cloning efficiency of the test cells, presumably owing to contact inhibition of growth and depletion of essential nutrients by the feeder layer cells. Regarding the effect of the feeder layers made of human fibroblasts, there were no significant differences in population doubling levels; tissue origins of fibroblasts; or fibroblasts derived from normal individuals, patients with cancer, or with a genetically high familial incidence of cancer, hereditary adenomatosis of the colon and rectum. This study was supported by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture, Japan.     

A cobblestone cell isolated from the human omentum: the mesothelial cell; isolation,identification, and growth characteristics

Summary Normal human mesothelial cells (NHMC) were isolated from pieces of human omentum. The cell yield was approximately one million cells per square centimeter omentum. The mesothelial cells were identified by their positive staining with monoclonal antibodies against cytokeratins 6 and 18. Transmission electronmicroscopy of cultured NHMC revealed many microvilli on the apical surface and many mitochondria and pinocytotic vesicles in the cytoplasm, indicating active transmembrane transport. Growth of NHMC was directly related to the concentration of human serum or of fetal bovine serum in the growth medium. Addition of epidermal growth factor with or without hydrocortisone resulted in a significant increase of NHMC growth; when endothelial cell growth factor, insulin, or hydrocortisone were added no such increase was observed. Seeding NHMC at densities less than 3000/cm² did not result in monolayer formation. The mesothelial cells were serially passed in growth medium M199 with added 10% fetal bovine serum up to 7 passages. However, after Passage 4 the cells changed into giant cells with an irregular pattern, and a lack of intracellular cytokeratin expression was observed for most of the cells.     

A retrovirus-producing transformed mouse cell line derived from a human breast adenocarcinoma transplanted in a nude mouse

Summary A transplantable tumor was established in NIH/Swiss/Nu mice from tissue derived from a human breast adenocarcinoma metastatic to the brain. Cultivation of dispersed cells from the third transplant generation of the tumor produced a rapidly growing, high-density culture of fibroblastlike cells. Chromosome and isozyme assays showed these cells to be of mouse origin. The cells behaved as an established line from initial culture. Cells of the tissue culture line, designated NM-1, produced rapidly growing fibrohistiocytomas in nude mice. Electron microscopy revealed that the cells produced large numbers of type C virus particles. Serological, biochemical, and infectivity assays indicated that the retrovirus produced by NM-1 cells is an ecotropic, infective, murine retrovirus antigenically related to, but distinguishable from, Gross and Moloney viruses. The virus did not transform mouse fibroblasts. The data support the conclusion that mouse stromal cells within the transplanted human tumor had undergone malignant transformation and induction to virus replication. The role of the virus in the malignant transformation remains to be clarified.     

Growth control of normal and transformed cells

Both serum factors and protein synthesis are required for normal cell growth. Swiss 3T3 cells require the serum growth factors insulin and EGF (epidermal growth factor) during the initial part of the G₁ period, until they pass a restriction point about 2 h before the initiation of DNA synthesis. Concentration of cycloheximide that inhibit protein synthesis by as much as 70% dramatically lengthen the cell cycle before the restriction point, while the cell cycle after the restriction point remains nearly constant. These results are consistent with a model in which labile proteins are required for transit of cells past the serum-sensitive restriction point. The relation of these findings to the growth control of transformed cells is discussed.     

Chemical and immunological studies of cell surfaces from normal and transformed cells

Immunological and chemical studies of cell surfaces from normal and transformed BALB/c fibroblasts have shown alterations associated with transformation. The cells studied include normal lines which do not cause tumors when injected into BALB/c mice, viral transformants, and spontaneous transformants which cause tumors that either regress or grow progressively, killing the host. The spontaneously transformed progressors include cell lines which are immunogenic and nonimmunogenic as determined by the ability of tumor excision to protect an animal from subsequent rechallenge by tumor cells. Tumor-bearing mice produce lymphocytes which are nonspecifically cytotoxic for all the normal and transformed lines. Some of the cell lines induce specific antibody formation in BALB/c hosts. Antisera have been prepared in rabbits which are specific for the transformed cell lines. These antisera can be used to determine specific surface changes on the transformed cells. Chemical studies have shown glycolipid alterations between the normal cells and some, but not all, of the transformants. Glycoproteins labeled by lactoperoxidase-¹²⁵ I or [³H] glucosamine were compared by SDS gel electrophoresis. Results from these studies do not show changes associated with malignancy. Individual glycoprotein regions from gels were treated with pronase, and the glycopeptides compared by Sephadex G-50 chromatography. Alterations in glycopeptides from several cellular glycoproteins are the only changes which appear to be associated with malignancy.     

Effects of establishing cell cultures and cell culture conditions on the proliferative life span of human fibroblasts isolated from different tissues and donors of different ages

It is well known that normal human cells placed in a culture environment exhibit a limited proliferative capacity. The extent to which the culture environment influences proliferative life span is not understood. This study evaluated the effects of the standard procedures used to establish and maintain cultures on the proliferative life spans of different types of human fibroblast cells established from fetal and adult skin and lung. The results of this study demonstrate that procedures to establish cell cultures use only one of several subpopulations of cells present in biopsy pieces and that the culture conditions routinely employed by most laboratories can exert significant effects on proliferative life-span determinations. The maximum proliferative life span differed significantly when obtained by growing the cells in two commonly used commercial media. Proliferative life span was inversely related to ambient oxygen tension and directly related to seeding density in all of the lines examined although lines established from adult skin were much more resistant to toxicity. Enzymatic antioxidant defense levels of fetal skin fibroblasts were much lower than those observed in adult skin fibroblasts, but the effects of oxygen on their life spans were similar. Hyperoxia induced larger increases in glutathione concentration in cell lines with low antioxidant enzyme levels.     

Factor(s) required by EBV transformed lymphocytes to grow under limiting dilution conditions

IL-6 was demonstrated to promote growth of EBV transformed lymphocytes. However IL-6 was ineffective at promoting growth of EBV transformed lymphocytes cultured at the single cell level under limiting dilution conditions. On the contrary, HECS, which is known to contain IL-6, supported very efficiently the growth of 1–2 EBV transformed cells. When IL-6 was removed from HECS, by using specific antibodies, no reduction in HECS activity was observed, indicating that probably more than one growth factor is required to support the growth of EBV transformed cells cultured at very low cell numbers in the absence of feeder cells.Abbreviations EBV Epstein Barr virus - FCS Foetal calf serum - HECS Human endothelial culture supernatant - IL-6 Interleukin 6 - PBL Peripheral blood lymphocytes - PBS Phosphate buffered saline     

Novel Madin Darby Canine Kidney cell clones exhibit unique phenotypes in response to morphogens

Summary Novel Madin Darby Canine Kidney cell clones were isolated. These cell clones exhibit differential responsiveness to inducers of tubule or cyst formation in collagen gel culture: hepatocyte growth factor or inducers of intracellular cAMP formation, respectively. In gel culture, clone OR93.22.D6 forms cysts and responds with morphological transformation to both hepatocyte growth factor and prostaglandin E₁, and is most typical of a previously described cell type except for its higher transepithelial electrical resistance. OR55.25.II20 forms tubules in culture, is unresponsive to hepatocyte growth factor, and forms prostaglandin-induced spherical cysts. OR55.28.V2 forms dense cell spheres under control conditions, is induced to form tubules by hepatocyte growth factor, and is unresponsive to prostaglandin. OR55.29 forms only cysts, and is the only clone to form domes in monolayer culture. Tubule formation induced by hepatocyte growth factor, in all clones except OR55.25.II20, is blocked by a neutralizing antibody. In defined medium, without hepatocyte growth factor or prostaglandin, OR55.25.II20 forms spontaneous tubules. This finding indicates that a tubulogenic serum factor is not responsible for the observed phenotype. Increasing prostaglandin concentrations lead to inhibition of tubule formation and increased cyst formation. This observation suggests that induction of intracellular cAMP formation negatively regulates tubule formation in these cells, and implies that cystogenesis may represent a “default pathway” for impaired tubulogenesis. These observations demonstrate that some facets of renal tubulogenesis may be independent of hepatocyte growth factor, and that care must be exercised when comparing biological studies utilizing different clones.     

Enzymatic characteristics of pp60v-src isolated from vanadium-treated transformed cells

The transforming protein of Rous sarcoma virus (RSV) typically appears as a single phosphorylated polypeptide designated pp60^v-src, pp60^v-src possesses a protein kinase activity specific for tyrosine residues on select protein substrates. Treatment of RSV-transformed cells with vanadium ions resulted in the appearance of an electrophoretic variant of pp60^v-src and was paralleled by a significant increase in the src kinase specific activity in purified enzyme preparations. Both the normal (standard) src kinase and the src kinase preparations obtained from vanadium-treated cells exhibited similar optimal activity profiles for MgCl₂, KCl, and pH. Furthermore, their site specificities of phosphorylation of the substrates casein and vinculin were the same. The reaction kinetic profile of the standard src kinase showed a nonlinear pattern, while the vanadium enzyme exhibited conventional linear Michaelis-Menten kinetics. These results are discussed with respect to the possible functional regulation of pp60^v-src activity by a vanadium-sensitive protein phosphatase activity.     

Effects of some antibiotics on the growth of human diploid skin fibroblasts in cell culture

Summary During serial subcultures 50 μg per ml gentamicin and penicillin (100 U per ml)-streptomycin (100 μg per ml) depressed cell growth significantly 2 weeks after the addition of the antibiotics; gentamicin, but not penicillin-streptomycin, stimulated cell growth before it became inhibitory. Removal of the antibiotics resulted in the cell yield returning to normal. The results show that these antibiotics can be harmful to cells even at concentrations thought to be safe.     

Platelets induce reactive oxygen species-dependent growth of human skin fibroblasts

A growing amount of evidence suggests that reactive oxygen species (ROS), such as hydrogen peroxide and superoxide anion, regulate intracellular signalling and have a role in cell proliferation. In the present study, we show that platelets increase the mitogenic rate in human fibroblasts and that this effect was inhibited by the intracellular antioxidant N-acetyl-L-cysteine (NAC) and the NADPH-oxidase inhibitor diphenyleneiodonium chloride (DPI). The mitogenic effects of platelets were mimicked by the platelet factors platelet-derived growth factor BB-isoform (PDGF-BB), transforming growth factor beta1 (TGF-beta1) and sphingosine-1-phosphate (S1P). The sphingosine kinase inhibitor DL-threo-dihydrosphingosine (DL-dihydro) abrogated the platelet-induced growth, while antibodies directed against PDGF or TGF-beta had modest effects. Exposure of fibroblasts to platelets, PDGF-BB, TGF-beta1 or S1P caused an extensive intracellular ROS production, measured as changes in dichlorofluorescein fluorescence. This ROS production was totally inhibited by NAC, pyrrolidinethiocarbamate (PDTC), DPI and apocynin. In conclusion, the results presented are indicative of a crucial role of ROS in the platelet-mediated regulation of fibroblast proliferation.     

Growth suppression of human transformed cells by treatment with bark extracts from a medicinal plant, Terminalia arjuna

Summary We have investigated the effects of acetone and methanol extracts of a medicinal plant, Terminalia arjuna, on the growth of human normal fibroblasts (WI-38), osteosarcoma (U2OS), and glioblastoma (U251) cells in vitro. We found that both extracts at 30 μg and 60 μg/ml concentrations inhibit the growth of transformed cells; the growth of normal cells was least affected. Although the transformed cells appeared to have fragmented nucleus by Hoechst staining, no deoxyribonucleic acid laddering effect was observed. In response to the extract treatment, the tumor suppressor protein, p53, was induced in U2OS but not in U251 and WI-38 cells. A cyclin-dependent kinase inhibitor, p21^WAF1, was induced in transformed cells only. The study suggests that the bark extract of medicinal plant, T. arjuna, has components that can induce growth arrest of transformed cells by p53-dependent and-independent pathways.     

Neuronal protein gene product 9.5 (IEF SSP 6104) is expressed in cultured human MRC-5 fibroblasts of normal origin and is strongly down-regulated in their SV40 transformed counterparts

Neuronal protein gene product 9.5 (PGP 9.5) most likely identical to ubiquitin carboxyl-terminal hydrolase isozyme LI (UCH-LI) has been reported to be expressed almost exclusively in neuronal and neuroendocrine tissues. By two-dimensional (2D) immunoblotting, comigration and microsequencing of proteins recovered from 2D gels we have identified PGP 9.5/UCH-LI as polypeptide IEF SSP 6104 (M_r = 27000, PL = 5.49) in the comprehensive 2D gel cellular protein database of human embryonal lung MRC-5 fibroblasts [(1989) Electrophoresis 10, 76–115; (1990) Electrophoresis 11, 1072–1113]. This protein is expressed at high levels in quiescent and proliferating cultured normal fibroblasts and is strongly down-regulated (about 10 times) in their transformed counterparts.     

Calcium,magnesium, and serum factors in multiplication of normal and transformed human lung fibroblasts

Summary Serum factors determine the extracellular requirement for both Ca²⁺ and Mg²⁺ for multiplication of normal human lung fibroblasts in vitro. Serum factors also affect the extracellular Ca²⁺ requirement for transformed fibroblasts but to a different extent than for normal cells. Transformed cells exhibit a reduced requirement for both Ca²⁺ and Mg²⁺ for multiplication. The apparent reduction in Ca²⁺ requirement of transformed cells is dependent on the level of serum factors in the medium. The reduced Mg²⁺ requirement for transformed cells is more striking than the loss of Ca²⁺ and independent of the level of serum factors in the medium. A sequential effector relationship among serum factors, Ca²⁺ and Mg²⁺, in a proliferative control system for normal cells is proposed. Alteration or bypass of an intracellular Mg²⁺-requiring process is proposed as a major lesion in the transformed cells. This alteration causes an observed loss of requirements for both Ca²⁺ and serum factors for the multiplication of transformed cells. This work was supported by Grant CA-15305 from the National Cancer Institute, Contract 223-74-1156 from the Bureau of Biologics, Food and Drug Administration, HEW Biomedical Research Support Grant S07RR05800, and the W. Alton Jones Foundation.     

Direct analysis of growth factor requirements for isolated human fetal hepatocytes

Summary Hepatocytes were isolated from human fetal liver in order to analyze the direct effects of growth factors and hormones on human hepatocyte proliferation and function. Mechanical fragmentation and then dissociation of fetal liver tissue with a collagenase/dispase mixture resulted in high yield and viability of hepatocytes. Hepatocytes were selected in arginine-free, ornithine-supplemented medium and defined by morphology, albumin production and ornithine uptake into cellular protein. A screen of over twenty growth factors, hormones, mitogenic agents and crude organ and cell extracts for effect on the stimulation of hepatocyte growth revealed that EGF, insulin, dexamethasone, and factors concentrated in bovine neural extract and hepatoma cell-conditioned medium supported attachment, maintenance and growth of hepatocytes on a collagen-coated substratum. The population of cells selected and defined as differentiated hepatocytes had a proliferative potential of about 4 cumulative population doublings. EGF and insulin synergistically stimulated DNA synthesis in the absence of other hormones and growth factors. Although neural extracts enhanced hepatocyte number, no effect on DNA synthesis of neural extracts or purified heparin-binding growth factors from neural extracts could be demonstrated in the absence or presence of defined hormones, hepatoma-conditioned medium or serum. Hepatoma cell-conditioned medium had the largest impact on both hepatocyte cell number and DNA synthesis under all conditions. Dialyzed serum protein (1 mg/ml) at 10 times higher protein concentration had a similar effect to hepatoma cell-conditioned medium (100 μg/ml). The results suggest that hepatoma cell conditioned medium may be a concentrated and less complicated source than serum for purification and characterization of additional normal hepatocyte growth factors. This work was supported by NIH grant DK35310. Editor’s statement Many investigators have struggled with the special problems associated with culture of differentiated hepatocytes. In this paper attention is given to the specific growth factor requirements for fetal human hepatocytes. The observation that factors from hepatoma conditioned medium or neural extracts enhanced the growth of the cells may indicate that additional growth factors are to be identified that are important in the survival and proliferation of hepatocytes, and may also indicate that the malignant transformation of these cells may involve the production of autocrine growth stimulators.     

High glucose-induced growth factor resistance in human fibroblasts can be reversed by antioxidants and protein kinase C-inhibitors

We have studied the influence of high glucose on basal fibroblast proliferation, growth factor induced cellular proliferation and the effects of antioxidants, protein kinase C-inhibitors and troglitazone. Fibroblast cultures were obtained from five patients undergoing mammary reduction plastic surgery. A fluorometric method was used for determining total DNA in the cell samples, DNA content being proportional to cell number. D -Glucose at 15·5 mM and above was shown to inhibit fibroblast proliferation, and the cells were resistant to growth factors such as IGF-I and EGF at this glucose concentration. H7, bisindolylmaleimide IX, troglitazone, α-tocopherol acetate, Q10, ascorbic acid, β-carotene, DMTU and selenite were all found to reverse the high glucose-induced growth factor resistance observed in human fibroblasts. We believe that these findings may be of value in the understanding and future treatment of wound healing in diabetic foot ulcers. © 1997 John Wiley & Sons, Ltd.     

A beta-type transforming growth factor, present in conditioned cell culture medium independent of cell transformation, may derive from serum

An alpha-type transforming growth factor (TGF alpha) is produced at high levels by rat embryo cells transformed by the Snyder-Theilen strain of feline sarcoma virus (FeSV). Addition of 2 ng mouse epidermal growth factor (mEGF) during purification identified the presence of a second, EGF-dependent growth factor of the TGF beta type (TGF beta) in this conditioned medium. This factor had an approximate Mr of 12,000 and eluted at 37% acetonitrile during high performance liquid chromatography. This extracellular type of TGF beta activity also was present in conditioned medium of rat cells after infection with a transformation defective strain of Abelson leukemia virus, and hence expression of this growth factor activity was independent of cell transformation. Moreover, the presence of an EGF-dependent, 12,000 Mr clonogenic activity in extracts of bovine serum alone suggests serum as an origin for the B-type transforming growth factor initially observed in conditioned medium of Snyder-Theilen FeSV transformed cells. This does not, however, preclude the possibility that TGF beta is also secreted by the transformed rat embryo cells themselves.