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.     

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.     

Recombinant human tumor necrosis factors alpha and beta stimulate fibroblasts to produce hemopoietic growth factors in vitro

The influences of TNF alpha and TNF beta were evaluated for their stimulatory and inhibitory effects on in vitro colony formation by human bone marrow granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells. Both TNF alpha and TNF beta induced fibroblasts to produce stimulators of CFU-GM, BFU-E, and CFU-GEMM in a dose-dependent fashion. Similar results were seen when equivalent concentrations of TNF alpha and TNF beta were used. Prior incubation of the TNF alpha and TNF beta with their respective antibodies inactivated the ability of the TNF preparations to induce the release of granulocyte-macrophage, erythroid, and multipotential colony-stimulating activity from fibroblasts. In addition, incubation of the TNF-induced fibroblast supernatant with antibody before colony assay resulted in enhanced colony formation, suggesting that the TNF carried over into the colony assay suppressed colony formation. Additional proof of this suppression by TNF was evident when TNF was added directly to the CFU-GM, BFU-E, and CFU-GEMM colony assays. IL-1 does not appear to function as an intermediary in growth factor production by fibroblasts stimulated with TNF because antibody to IL-1 displayed no effect. Furthermore, assay of TNF-induced fibroblast supernatant was negative for IL-1. These results suggest that TNF alpha and TNF beta exert both a positive and negative influence on in vitro hemopoietic colony formation.     

Growth of normal human keratinocytes and fibroblasts in serum-free medium is stimulated by acidic and basic fibroblast growth factor

Keratinocytes and fibroblasts isolated from human neonatal foreskin can be plated and grown through multiple rounds of division in vitro under defined serum-free conditions. We utilized these growth conditions to examine the mitogenic potential of acidic and basic fibroblast growth factor (aFGF and bFGF) on these cells. Our results demonstrate that both aFGF and bFGF can stimulate the proliferation of keratinocytes and fibroblasts. aFGF is a more potent mitogen than bFGF for keratinocytes. In contrast, bFGF appears to be more potent than aFGF in stimulating the growth of fibroblast cultures. Heparin sulfate (10 micrograms/ml) dramatically inhibited the ability of bFGF to stimulate the proliferation of keratinocytes. In comparison, heparin slightly inhibited the stimulatory effect of aFGF and had no effect on epidermal growth factor (EGF) stimulation in keratinocyte cultures. In fibroblast cultures the addition of heparin enhanced the mitogenic effect of aFGF, had a minimal stimulatory effect on the mitogenic activity of bFGF, and had no effect on EGF-stimulated growth. Our results demonstrate that the proliferation in vitro of two normal cell types found in the skin can be influenced by aFGF and bFGF and demonstrate cell-type specific differences in the responsiveness of fibroblasts and keratinocytes to these growth factors and heparin.     

Modulation of growth and differentiation in normal human keratinocytes by transforming growth factor-beta

The effect of transforming growth factor-type beta 1(TGF-beta) on the growth and differentiation of normal human skin keratinocytes cultured in serum-free medium was investigated. TGF-beta markedly inhibited the growth of keratinocytes at the concentrations greater than 2 ng/ml under low Ca2+ conditions (0.1 mM). Growth inhibition was accompanied by changes in cell functions related to proliferation. Remarkable inhibition of DNA synthesis was demonstrated by the decrease of [3H]thymidine incorporation. The decrease of [3H]thymidine incorporation was observed as early as 3 hr after addition of TGF-beta. TGF-beta also decreased c-myc messenger RNA (mRNA) expression 30 min after addition of TGF-beta. This rapid reduction of c-myc mRNA expression by TGF-beta treatment is possibly one of the main factors in the process of TGF-beta-induced growth inhibition of human keratinocytes. Since growth inhibition and induction of differentiation are closely related in human keratinocytes, the growth-inhibitory effect of TGF-beta under high Ca2+ conditions (1.8 mM Ca2+, differentiation-promoting culture environment) was examined. TGF-beta inhibited the growth of keratinocytes under high Ca2+ conditions in the same manner as under low Ca2+ conditions, suggesting that it is a strong growth inhibitor in both low and high Ca2+ environments. The induction of keratinocyte differentiation was evaluated by measuring involucrin expression and cornified envelope formation: TGF-beta at 20 ng/ml increased involucrin expression from 9.3% to 18.8% under high Ca2+ conditions, while it decreased involucrin expression from 7.0% to 3.3% under low Ca2+ conditions. Cornified envelope formation was modulated in a similar way by addition of TGF-beta: TGF-beta at 20 ng/ml decreased cornified envelope formation by 53% under low Ca2+ conditions, while it enhanced cornified envelope formation by 30.7% under high Ca2+ conditions. Thus, the effect of TGF-beta on keratinocyte differentiation is Ca2+ dependent. It enhances differentiation of human keratinocytes under high Ca2+ conditions, but inhibits differentiation under low Ca2+ conditions. Taken together, there is a clear discrepancy between TGF-beta effects on growth inhibition and induction of differentiation in human keratinocytes. These data indicate that growth inhibition of human keratinocytes by TGF-beta is direct and not induced by differentiation.     

The non-selective junctional communication phenotype of normal and transformed human epidermal keratinocytes in vitro

The ability of cells to transfer tritium-labelled nucleotides to other cells is a measure of gap-junction-mediated communication based on metabolic cooperation. Here we report that human epidermal keratinocytes (HuK) transfer radiolabel to a variety of cell types including human skin fibroblasts (HuDF), human mammary fibroblasts (HuMF) and calf lens epithelial cells (CaLE). The metabolic cooperation pattern of HuK shows them to be non-selective communicators and in this respect they differ from another human epithelial cell type mammary epithelial cells (HuME), which communicates with CaLE but not HuDF or HuMF. SV40 transformation is known to modulate metabolic cooperation between some human cells in vitro. Here we further report that SV40 transformation of HuK has no obvious effect on their non-selective communication phenotype. Possible mechanisms involved in selective and non-selective junctional communication and their relevance to epithelial/stromal interactions in vivo are discussed.     

Low calcium culture condition induces mesenchymal cell-like phenotype in normal human epidermal keratinocytes

Epithelial–mesenchymal transition (EMT) is an important cellular phenomenon in organ developments, cancer invasions, and wound healing, and many types of transformed cell lines are used for investigating for molecular mechanisms of EMT. However, there are few reports for EMT in normal human epithelial cells, which are non-transformed or non-immortalized cells, in vitro. Therefore, normal human epidermal keratinocytes (NHEK) serially cultured in low-calcium concentration medium (LCM) were used for investigating relations between differentiation and proliferation and mesenchymal-like phenotype in the present study, since long-term cultivation of NHEK is achieved in LCM. Interestingly, NHEK serially cultured in LCM consisted essentially of cytokeratin–vimentin double positive cells (98%), although the NHEK exhibited differentiation under high-calcium culture condition with 3T3 feeder layer. The vimentin expression was suppressed under high-calcium condition. These results may indicate the importance of mesenchymal-like phenotype for serially cultivation of NHEK in vitro.     

Bone marrow stromal cells produce nerve growth factor and glial cell line-derived neurotrophic factors

Bone marrow stromal cells (BMSC) have attracted interest through their possible use for cell therapy in neurological diseases. Recent reports demonstrated that these cells are able to migrate and have potential for neuronal differentiation after transplantation into brain parenchyma. The objective of this work was determine whether rat BMSC express NGF and GDNF, in order to study its potential application for treatment of neurodegenerative diseases. BMSC were harvested from male rats and cultured in DMEM supplemented with 20% fetal bovine serum. At passage 6 the total RNA was isolated using TriZol reactive. RT-PCRs to evaluate the expression of NGF and GDNF using specific primers were carried out. Our results indicate that rat BMSC have potential to produce NGF and GDNF. We have not found any report in favor of GDNF or NGF production from rat BMSC.     

Contraction of collagen by human fibroblasts and keratinocytes

Summary In the process of wound healing keratinocytes and fibroblasts play an important role, keratinocytes in the re-epithelization process and fibroblasts in the process of wound contraction. We have studied the role of human keratinocytes and fibroblasts in the rearrangement of collagen in a collagen lattice model system. Our results revealed that keratinocytes as well as fibroblasts rearrange the collagen lattice; this occurs in a cell number and collagen concentration dependent manner. The optimal gel contraction is obtained in the presence of keratinocytes on the top of and of fibroblasts in the collagen lattice, the situation most closely approaching the in vivo situation. Between the two types of cells, differences in morphologic behavior were observed: when incorporated into the gel the keratinocytes retained their spherical shape throughout the whole culture period, but fibroblasts became elongated and formed extensions. Our data suggest that not only fibroblasts but also keratinocytes may be involved in the process of wound contraction. This work was supported by the Koningin Wilhelmina Fonds (Netherlands Cancer Foundation, grant 84-10).     

Clonal growth of normal human epidermal keratinocytes in a defined medium

Early studies on the duration of mitotic stages and on metaphase-to-prophase ratios in a number of normal and neoplastic cells indicated that the process of mitosis becomes altered during the course of oncogenesis. However, the nature of these changes and their effects on each of the mitotic stages are still unclear. With the use of time lapse cinemicrography, we have compared the durations of mitotic stages of SV40/Wl-38 and SV40/Wl-26 cells to those of their normal counterparts and to other nontransformed human fibroblasts. We also examined the relative frequencies of the individual mitotic stages in fixed preparations of Wl-38 and SV40/Wl-38 cells. The data show that metaphase durations are increased in the transformed cells and as much as 3-4.7-fold in SV40/Wl-38 cells compared to Wl-38 and other nontransformed cells. Other stages are also prolonged though to lesser degrees. These findings suggest that increased metaphase/prophase ratios observed in many tumors are due to increases in duration of metaphase rather than to shorter prophases, and that increased mitotic indices commonly observed in malignant tumors and sometimes used as an index of growth rate are at least in part due to the prolongation of mitotic stages.     

Head and neck squamous cell carcinoma: role of the human papillomavirus in tumour progression

High risk human papilloma viruses (HPVs) have been shown to be independent risk factors for anogenital tract cancers, and have also been detected in head and neck squamous cell carcinomas (HNSCC). The aim of our study was to determine the prevalence of HPV DNA in a group of 47 squamous cell carcinomas of the oropharynx and the oral cavity, and to compare the clinical behaviour of HPV positive and negative tumours. We also assessed the proliferation index, as evaluated by Ki67 immunohistochemistry positivity, and the level of p53 reactivity. HPV DNA was found in 50% of carcinomas of the oropharynx and 36% in those of the oral cavity, the only genotype detected being HPV 16. Patients with HPV-positive carcinomas had a better overall survival than those with HPV-negative carcinomas. Our data suggest that HPV-positive oropharyngeal cancers comprise a distinct disease entity with an improved prognosis.     

Interleukin-1 regulation of hematopoietic growth factor production by human stromal fibroblasts

The human stromal fibroblastoid cell strain designated ST-1 represents a normal population of cells capable of supporting hematopoiesis in vitro. These cells constitutively elaborate hematopoietic growth factor activity into the medium and the level of production of this activity dramatically increases following stimulation of the cells with IL-1. This enhanced production is due at least in part to increased expression of the genes for GM-CSF, G-CSF, and IL-6, but not IL-3. The IL-1 treatment had little effect on the expression of M-CSF, a factor made constitutively by the cells. These results are consistent with the model that hematopoiesis is regulated at least in part by constant short-range interactions of humoral factors produced by stromal cells both with other types of stromal cells and with the hematopoietic progenitors.     

Proliferation and differnetiation of human squamous carcinoma cell lines and normal keratinocytes: Effects of epidermal growth factor,retinoids, and hydrocortisone

Summary Exposure of squamous carcinoma cell (SCC) lines, exhibiting high levels of epidermal growth factor (EGF) receptors, to EGF for 6 d caused a dose-dependent inhibition of cell proliferation. This EGF-induced inhibition of cell proliferation occurred under both low (0.06 mM) and normal (1.6 mM) Ca²⁺ concentrations. Furthermore, the extent of EGF-induced inhibition of cell proliferation seemed to be independent of the number of EGF-receptors. This conclusion is based on the notion that the various SCC lines exhibited an increasing number of EGF receptors accompanied by a decreasing ability to differentiate, whereas no relationship was observed with the EGF-induced inhibition of cell proliferation in these cell lines. Retinoids caused also a dose-dependent inhibition of cell proliferation. The effects of EGF and retinoids were additive, indicating that different regulatory mechanisms are involved. On the other hand, hydrocortisone caused a stimulation of SCC-proliferation, also independent of EGF. In contrast to SCC cells, EGF did not affect significantly the rate of proliferation of normal keratinocytes. However, the simultaneous addition of EGF and hydrocortisone resulted in a significant increase in the rate of keratinocyte proliferation only in cells grown under normal calcium conditions. Differentiation capacity of normal keratinocytes and SCC lines was not affected by EGF. Furthermore, the retinoid-induced decrease and hydrocortisone-induced increase of competence of cells to form cornified envelopes was not affected by EGF. These observations suggest that the action of retinoids and hydrocortisone on both cell proliferation and cell differentiation occurs independently of EGF receptors. This work was partly supported by The Netherlands Cancer Foundation (Koningin Wilhelmina Fonds), grant IKW 85–71.     

Effect of growth factors on morphogenesis of human keratinocytes in vitro

The effect of some growth factors on morphogenetic processes in the primary culture of human epidermal keratinocytes was studied in the model of formation of tubule-like structures in collagen gel. Culturing of keratinocytes in the presence of IGF and bFGF did not induce growth of tubule-like structures, whereas EGF, KGF, and HGF promoted the growth of three-dimensional epidermal outgrowths whose shape and size varied depending on the growth factor used.     

UVB radiation affects the mobility of epidermal growth factor receptors in human keratinocytes and fibroblasts

Growth factor receptors transmit biological signals for the stimulation of cell growth in vitro and in vivo and their autocrine stimulation may be involved in tumorigenesis. It is therefore, of great value to understand receptor reactions in response to ultraviolet (UV) light which certain normal human cells are invaribly exposed to during their growth cycle. UV irradiation has recently been shown to deplete antioxidant enzymes in human skin. The aims of the present study were a) to compare the lateral mobility of epidermal growth factor receptors (EGF-R) in cultured human keratinocytes and human foreskin fibroblasts, b) to investigate effects of ultraviolet B radiation on the mobility of EGF-R in these cells, and c) study the response of EGF-R on addition of antioxidant enzymes. The epidermal growth factor receptors were labeled with rhodaminated EGF, the lateral diffusion was determined and the fraction of mobile EGF-R assessed with the fluorescence recovery after photobleaching (FRAP). We found that human keratinocytes display a higher basal level of EGF-R mobility than human skin fibroblasts, viz. with diffusion coefficients (D ± standard error of the mean, SEM) of 4.2±0.2 × 10^–10 cm²/s, and 1.8±0.2 × 10^–10 cm²/s, respectively. UVB-irradiated fibroblasts showed an almost four-fold increase in the diffusion coefficient; D was 6.3±0.3 × 10^–10 cm²/s. The keratinocytes, however, displayed no significant increase in receptor diffusion after irradiation; D was 5.1±0.8 × 10^–10 cm²/s. In both cell types the percentage of EGF-R fluorescence recovery after photobleaching, i.e. the fraction of mobile receptors, was significantly increased after irradiation. In keratinocytes it increased from 69% before irradiation to 78% after irradiation. Analogous figures for fibroblasts were 61% and 73%. The effect of UVB on fibroblast receptors was abolished by prior addition of superoxide dismutase (SOD) and catalase (CAT). It is concluded that UVB radiation of fibroblasts and keratinocytes can affect their biophysical properties of EGF-R. The finding that addition of antioxidant enzymes prevented the UVB effect in fibroblasts may indicate the involvement of reactive oxygen metabolites.Abbreviations CAT Catalase - D Lateral diffusion coefficient - EDTA Ethylenediaminetetraacetic acid - EGF Epidermal growth factor - E-MEM Eagle's minimum essential medium - FCS Fetal calf serum - FRAP Fluorescence recovery after photobleaching - KRG Krebs-Ringer phosphate buffer - PBS Phosphate-buffered saline - R Mobile fraction - ROS Reactive oxygen species - SEM Standard error of the mean - SOD Superoxide dismutase - UVA Ultraviolet light-A (315-400 nm) - UVB Ultraviolet light-B (280-315 nm)     

Endothelin-1 acts as an autocrine growth factor for normal human keratinocytes

Colorectal cancers are often composed of cell types representing various differentiated cell lineages, however little is known concerning the relationship of differentiation and drug resistance in these cancers. The present study was performed to develop and characterize a stable, differentiated clone of the human colon cancer cell line LS174T and to characterize the drug resistance of this cell line in relation to its undifferentiated parental cell line. LS174T cell line was treated with the differentiating agent sodium butyrate (0.5 mM) for 30 days, then recultured in standard medium. Foci of flat-appearing cells appeared and were isolated using cloning rings, and subcloned. One subclone was designated LS174T-D. The LS174T-D clone maintains a stable, differentiated phenotype in standard culture conditions in the absence of sodium butyrate. It is characterized by the formation of a polarized monolayer with dome formation and the presence of prominent apical microvilli and tight junctions. This cell line demonstrated reduced growth in soft agar and nude mice compared with the parental cell line. LS174T-D cells expressed immunoreactive intestinal mucin antigens and brush border enzymes dipeptidyl aminopeptidase (DAP)-IV and aminopeptidase. The activities of DAP-IV and aminopeptidase were increased 5.6-fold and 3.4-fold, respectively, in LS174T-D compared with parental cells. Proliferation assays demonstrated that, compared with the parental cell line, LS174T-D cells were more resistant to doxorubicin (93-fold), cisplatin (23-fold), 5-fluorouracil (12-fold), 5-fluorodeoxyuridine (31-fold), and methotrexate (12.5-fold). Intracellular uptake of (³H)-5-fluorodeoxyuridine did not differ significantly in the differentiated and undifferentiated cell lines. Levels of mdr-1 p-glycoprotein measured by Western blot and RNA Northern blot assays were also similarly low in both cell lines. However, total glutathione content and glutathione-S-transferase activities were increased in LS174T-D cells by sixfold and threefold, respectively, compared with parental cells. Depletion of glutathione by pretreatment with DL-buthionine sulfoximine reversed LS174T-D resistance to cisplatin. Long-term treatment with sodium butyrate induces or selects for colon cancer cells with features of enterocytic differentiation. This stably differentiated cell line is associated with glutathione-mediated multidrug resistance, and provides a model for further studies of differentiation in normal and cancerous colon. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America

     

Heterogeneity of cancer-associated fibroblasts in head and neck squamous cell carcinoma

Cancer-associated fibroblasts (CAFs) consist of heterogeneous cellular populations that contribute critical roles in head and neck squamous cell carcinoma (HNSCC). A series of computer-aided analyses were performed to determine various aspects of CAFs in HNSCC, including their cellular heterogeneity, prognostic value, relationship with immune suppression and immunotherapeutic response, intercellular communication, and metabolic activity. The prognostic significance of CKS2+ CAFs was verified using immunohistochemistry. Our findings revealed that fibroblasts group demonstrated prognostic significance, with the CKS2+ subset of inflammatory CAFs (iCAFs) exhibiting a significant correlation with unfavorable prognosis and being localized in close proximity to cancer cells. Patients with a high infiltration of CKS2+ CAFs had a poor overall survival rate. There is a negative correlation between CKS2+ iCAFs and cytotoxic CD8+ T cells and natural killer (NK) cells, while a positive correlation was found with exhausted CD8+ T cells. Additionally, patients in Cluster 3, characterized by a high proportion of CKS2+ iCAFs, and patients in Cluster 2, characterized by a high proportion of CKS2- iCAFs and CENPF-/MYLPF- myofibroblastic CAFs (myCAFs), did not exhibit significant immunotherapeutic responses. Moreover, close interactions was confirmed to exist between cancer cells and CKS2+ iCAFs/ CENPF+ myCAFs. Furthermore, CKS2+ iCAFs demonstrated the highest level of metabolic activity. In summary, our study enhances the understanding of the heterogeneity of CAFs and provided insights into improving the efficacy of immunotherapies and prognostic accuracy for HNSCC patients.     

Site-matched papillary and reticular human dermal fibroblasts differ in their release of specific growth factors/cytokines and in their interaction with keratinocytes

The interfollicular dermis of adult human skin is partitioned into histologically and physiologically distinct papillary and reticular zones. Each of these zones contains a unique population of fibroblasts that differ in respect to their proliferation kinetics, rates at which they contract type I collagen gels, and in their relative production of decorin and versican. Here, site-matched papillary and reticular dermal fibroblasts couples were compared to determine whether each population interacted with keratinocytes in an equivalent or different manner. Papillary and reticular fibroblasts grown in monolayer culture differed significantly from each other in their release of keratinocyte growth factor (KGF) and granulocyte-macrophage colony stimulating factor (GM-CSF) into culture medium. Some matched fibroblast couples also differed in their constitutive release of interleukin-6 (IL-6). Papillary fibroblasts produced a higher ratio of GM-CSF to KGF than did corresponding reticular fibroblasts. Interactions between site-matched papillary and reticular couples were also assayed in a three-dimensional culture system where fibroblasts and keratinocytes were randomly mixed, incorporated into type I collagen gels, and allowed to sort. Keratinocytes formed distinctive cellular masses in which the keratinocytes were organized such that the exterior most layer of cells exhibited characteristics of basal keratinocytes and the interior most cells exhibited characteristics of terminally differentiated keratinocytes. In the presence of papillary dermal fibroblasts, keratinocyte masses were highly symmetrical and cells expressed all levels of differentiation markers. In contrast, keratinocyte masses that formed in the presence of reticular fibroblasts tended to have irregular shapes, and terminal differentiation was suppressed. Furthermore, basement membrane formation was retarded in the presence of reticular cells. These studies indicate that site-matched papillary and reticular dermal fibroblasts qualitatively differ in their support of epidermal cells, with papillary cells interacting more effectively than corresponding reticular cells.