Selective inhibition of fibroblasts by spermine in primary cultures of normal human skin epithelial cells

Summary Overgrowth with fibroblasts has been a major problem in the cultivation of normal human skin epithelium. In the present study it is shown that the addition of spermine to the culture medium in micromolar concentrations has a differential cytotoxic effect on fibroblasts allowing the cultivation of human skin epithelial cells in primary culture without fibroblastic overgrowth. Putrescine, another polyamine, is shown to be equally cytotoxic to fibroblasts and epithelial cells when added in millimolar concentrations; below this concentration range no cytotoxic effect could be demonstrated. This difference in cytotoxicity between spermine and putrescine is suggested to depend on the conversion of spermine, but not putrescine, and to highly cytotoxic products by an amine oxidase present in fetal bovine serum. This project was supported by the Novo foundation.     

CD90 Expression on human primary cells and elimination of contaminating fibroblasts from cell cultures

Cluster Differentiation 90 (CD90) is a cell surface glycoprotein originally identified on mouse thymocytes. Although CD90 has been identified on a variety of stem cells and at varying levels in non-lymphoid tissues such as on fibroblasts, brain cells, and activated endothelial cells, the knowledge about the levels of CD90 expression on different cell types, including human primary cells, is limited. The goal of this study was to identify CD90 as a human primary cell biomarker and to develop an efficient and reliable method for eliminating unwanted or contaminating fibroblasts from human primary cell cultures suitable for research pursuant to cell based therapy technologies.     

Selective elimination of alleles in rice anther cultures

The nature of heterosis is discussed and selective elimination of alleles (introduced in the hybrid genotype by the parental forms) in anther culture is shown. This supports the possibility of removing viability-reducing alleles (lethal, semilethal, and less effective alleles) from the genotypes of heterotic hybrids in anther culture.     

Gangliosides of normal and neoplastic human melanocytes

The major ganglioside component isolated from diploid human melanocytes is sialosyllactosylceramide (GM3 86-91% of total sialic acid). The corresponding disialo derivative (GD3) is found as a minor component (2-6% of total sialic acid) in the membranes of these cells. In human melanoma cells, grown in tissue culture, GD3 is the predominant ganglioside component (48-63% of total sialic acid). Withdrawal of TPA from the culture medium of normal melanocytes or addition of TPA to the medium of melanoma cells had no significant effect on GM3/GD3 ratios. We conclude that the difference between the composition of gangliosides is related to the normal vs transformed phenotypes of melanocytes.     

Removal of glycosaminoglycans from cultures of human skin fibroblasts.

Early-passage human skin fibroblasts were grown as monolayers for 2-3 days in minimum essential medium containing [35S]sulphate, [3H]glucosamine, [3H]fucose, [3H]proline or [3H]leucine to label proteoglycans, glycoproteins or collagen and other proteins. A crude enzyme preparation obtained from a supernatant from sonicated freeze-dried Flavobacter heparinum was added to the cell monolayers. This treatment removed most of the 35S-labelled glycosaminoglycans, with no appreciable removal of the 3H-labelled proteins or 3H-labelled glycoproteins. The cells remained attached and viable as a monolayer. The formation of 35S-labelled glycosaminoglycans was examined after pretreating cultures with crude F. heparinum enzyme, followed by addition of fresh growth medium containing [35S]sulphate. The F. heparinum enzyme did not significantly alter the amount or type of 35S-labelled glycosaminoglycans produced. Thus F. heparinum enzyme can be used to provide cultured-cell monolayers depleted of surface glycosaminoglycans. These cells remain attached, viable and subsequently synthesize normal amounts and type of glycosaminoglycans.     

Spreading-independent growth of normal fibroblasts in three-dimensional cultures

Changes of cell shape resulting from cellular flattening on culture substratum have previously been demonstrated to correlate with mitotic activity of normal animal cells in monolayer cultures. Here, we compared the shapes and proliferation of chick embryo fibroblasts cultured either in multicellular, multilayered sheets extended between glass fibres, or in standard monolayers. Fibroblasts in sheets retained the mitotic activity characteristic of that observed in sparse monolayer cultures, i.e. considerably higher that in confluent monolayers. Morphometric analyses revealed, however, that the cells in sheets were considerably less flattened than in monolayer cultures. These observations indicate that the modulation of culture conditions resulting in multidirectional cell stretching leads to the dissociation of flattening and mitotic activity of normal animal cells, so long as an intracellular stress field, generated by contractile cytoskeleton and stabilised by intercellular contacts, is maintained.     

Spreading of normal and transformed fibroblasts in dense cultures

Cell spreading in dense cultures of normal mouse embryo fibroblasts and of the two lines of mouse transformed fibroblasts was examined by electron microscopy. The mean number of cell layers in culture and cell population density per unit area of the substrate were detetmined; the mean area of the cell projection on the substratum was found from these data.Normal fibroblasts formed multilayefed sheet in dense culture. The cells in this sheet were well-spread. These cells formed thin lamellae (lamellar cytoplasm) over the surface of other cells and over the intercellular substance. The mean cell area in dense culture was not smaller than that of the cell spread on the substratum in sparse culture.Dense cultures of two transformed lines (M 22 and L) had differing morphologies: cultures of one line (M 22) were multilayered, those of the other line (L) were monolayered. Decreased spreading and almost complete (M 22) or complete (L) absence of lamellar cytoplasm were characteristic of both transformed lines. The mean area of the cell in dense cultures of both lines was several times smaller than that of their normal progenitors.It is concluded that similar reactions leading to the spreading accompanied by the formation of lamellar cytoplasm can be induced by the contact of fibroblast with various surfaces: that of the substratum in sparse culture, that of other cells and of intercellular structures in dense culture. Deficiency of these reactions characteristic for transformed fibroblasts may be responsible for abnormal morphology of their cultures.     

Depigmenting effect of Xanthohumol from hop extract in MNT-1 human melanoma cells and normal human melanocytes

Xanthohumol (XH) is the most abundant prenylated flavonoid found in the hop plant (Humulus lupulus L.) and has previously been shown to have depigmenting effects in B16F10 mouse melanoma cells; however, studies of its depigmenting efficacy in human melanocytes are still lacking. In this work, we explored the effects of XH on melanogenesis in MNT-1 human melanoma cells and normal human melanocytes from darkly-pigmented skin (HEM-DP). XH was screened for cytotoxicity over 48 h, and subsequently tested on melanogenesis in MNT-1 cells. XH was further tested in HEM-DP cells for melanin synthesis and melanosome export; dendricity was quantitated to assess effects on melanosome export. Melanosome degradation was studied in human keratinocytes (HaCaT). Our results showed that XH inhibited melanin synthesis in MNT-1 cells at 30 μM but increased intracellular tyrosinase activity without affecting ROS levels. In HEM-DP cells, XH robustly suppressed cellular tyrosinase activity at nontoxic concentrations (2.5–5 μM) without any effect on melanin synthesis. However, XH inhibited melanosome export by reducing dendrite number and total dendrite length. Further testing in HaCaT cells demonstrated that XH induced melanosome degradation at low micromolar concentrations without any cytotoxicity. In summary, our results demonstrate that XH at low micromolar concentrations might hold promise as a potent inhibitor of human pigmentation by primarily targeting melanin export and melanin degradation. Further studies to elucidate the signaling mechanisms of action of melanosome export inhibition by XH and in vivo efficacy are warranted.     

Abnormal collagen in cultures of fibroblasts from human beings with diabetes mellitus

Synthesis of collagen by fibroblasts from diabetic human beings was investigated to determine if abnormal synthesis might be responsible for an apparent acceleration of collagen aging observed previously in diabetics. Increased material was synthesized by cells from diabetics, and the material was abnormal in that it contained a very high molecular weight material and lacked a presumed procollagen dimer. Alpha chains did not appear altered. It is suggested that cultures of cells from diabetics yield a non-helical peptide of procollagen that contains abnormal reducible cross links and lacks normal non-reducible cross links.     

Effects of human transforming growth factors on topoisomerases from normal fibroblasts

Normal rat kidney fibroblasts (NRK-B F49) treated at transforming doses with a gamma-like TGF, partially purified from human melanoma, showed a 3 to 5 fold increase in DNA type II topoisomerase activity. A similar effect was observed using EGF and a partially purified alfa TGF from rabbit fetuses. DNA type I topoisomerase activity, from the same cells, was not significantly modified by treatment with these growth factors. Topoisomerase II stimulation was dependent on mRNA synthesis. The possible role of topoisomerase II in phenotypical cell transformation induced by TGF is discussed.     

Primary cultures of hepatocytes on human fibroblasts.

Parenchymal hepatocytes isolated from adult rats were cultured on three types of collagen-containing substrata: collagen-coated plates, collagen membranes and confluent diploid human fibroblasts. Hepatocytes on the latter two substrata maintained characteristic morphology for at least 10 days in culture, whereas degenerative changes (cell death and formation of multinucleated hepatocytes) and growth of nonparenchymal elements were seen after 5 days in cultures on collagen-coated plates. Parallel findings were seen on basal and induced levels of cytochrome P-450 and NADPH-cytochrome C reductase. The basal levels of cytochrome P-450 were not measurable after day 3 in hepatocytes cultured on collagen-coated plates, whereas measurable levels were maintained in the hepatocytes cultured on the other two substrata. Addition of phenobarbital or methylcholanthrene at day 5 in culture caused an increase in cytochromes P-450 and P-448, respectively, only in hepatocytes cultured on collagen membranes and confluent fibroblasts. Analogous results were seen for the enzyme NADPH-cytochrome C reductase. The similarities in performance between hepatocytes on collagen membranes and on human fibroblasts show that a continuous collagen-containing substratum is important for optimal performance of hepatocytes in primary culture. The possible importance of cultures of hepatocytes on human fibroblasts for carcinogenesis studies is discussed.     

Collagen, proteoglycan and hyaluronidase activity in cultures from normal and scoliotic chicken fibroblasts

Connective tissue matrix components were investigated using skin fibroblasts from normal or inbred scoliotic lines of chickens. Specifically, the fibroblasts were obtained from either an isogenic line or a backcross, derived by crossing the isogenic line with a pure line of scoliotic birds. From the backcross, both affected (35-45%) and non-affected (55-65%) progeny were produced. The affected birds had spinal curves greater than 20 degrees. Several abnormalities of connective tissue were observed when cells from scoliotic chicks were grown in culture: increased collagen extractability, decreased aggregatability of proteoglycans under associative conditions and lower than normal levels of hyaluronic acid. There was also less collagen deposited in the cell layer with proportionately increased amounts of collagen secreted into the culture media by cells from scoliotic versus normal chick fibroblasts. Values for collagen matrix stability, as estimated by extractability and net deposition, were intermediate for cells from the backcrossed, but non-affected, birds. Moreover, hyaluronidase, an enzyme that degrades hyaluronic acid, was abnormally elevated in the fibroblast cultures from scoliotic chicks. It is proposed that the increase in hyaluronidase contributes to the abnormalities observed in extracellular matrix components and may be a factor in the expression of scoliosis in susceptible birds.     

Characteristics of long-term human epithelial cell cultures derived from normal human fetal kidney

Summary Epithelial cell cultures were prepared from normal human fetal kidney and established in long-term culture. The growth characteristics and production of keratin, and alkaline phosphatase (AP) and gamma-glutamyl transpeptidase (GGT) activities were compared in a modified minimal essential medium (mMEM),d-valine-containing modified alpha-MEM (mALPHA) andl-valine mALPHA. The mean number of cumulative population doublings (CPDL) was significantly (P<0.001) enhanced with thel-valine mALPHA (40.8 CPDL) over that achievable in mMEM (14.2 CPDL) ord-valine mALPHA (18.3 CPDL) media. In all three media, greater than 95% of the cells in culture produced keratin throughout the life span of these cultures. Surface-associated fibronectin was absent in these cell cultures. AP and GGT activities increased as a function of subpassage and time in culture, with the greatest activity in thel-valine mALPHA. The expression of these renal cell-associated functions suggests that these cells in culture are proximal tubule epithelial cells. The conditions and procedures described in this paper can provide a human kidney epithelial cell culture system for studying human renal function, metabolism, cytotoxicity, genotoxicity, and transformation. Research was supported by a NIEHS (ES 3101) grant to S. M. D’Ambrosio and a NCI grant (CA21104) to J. E. Trosko.     

Regulatory factors that determine growth and phenotype of normal human melanocytes

Normal human melanocytes, unlike malignant melanoma cells, required at least three growth-promoting agents, i.e., phorbol ester for protein kinase C activation and the growth factors basic fibroblast growth factor (bFGF) and insulin, for growth in chemically defined W489 medium. Cell growth was further stimulated by addition of agents that increase intracellular levels of cyclic adenosine 3',5'-monophosphate (cAMP) to the medium. Among these agents, the pituitary hormones alpha-melanocyte-stimulating hormone (alpha-MSH) and follicle-stimulating hormone were the most potent, whereas bacterial toxins, including cholera, tetanus, and pertussis toxin and their subunits either were less mitogenic or gave variable results depending on the culture tested. Medium containing phorbol ester PMA, growth factors bFGF and insulin (or insulin-like growth factor-I), and synthetic alpha-MSH supported melanocyte growth for more than 5 months with doubling times between 5 and 8 days. Two copper-binding proteins, ceruloplasmin and tyrosinase, were mitogenic when added to medium and ceruloplasmic induced a long bi- to tripolar-shape of cells. Addition of 1 mM dibutyryl cAMP to the medium led to the formation of dendrites in all cells, with an average of 28 extensions per cell. Although cell growth was inhibited by dibutyryl cAMP, cells were not terminally differentiated and continued to proliferate. Dendritic melanocytes showed a 2.2-fold increase in activity of the tyrosine kinase pp60c-src. The induction of dendritic processes in melanocytes by dibutyryl cAMP or sodium butyrate was reversible and appears to reflect the expression of the mature melanocytic phenotype in situ.