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E E Medrano

Publications and source records attributed to E E Medrano.

At least 37 records · Page 2Linked to original sources
PubMed1995

Long-term proliferation of human melanocytes is supported by the physiologic mitogens alpha-melanotropin, endothelin-1, and basic fibroblast growth factor.

We have successfully established normal neonatal and adult human melanocyte cultures in a growth medium containing the physiologic mitogens basic fibroblast growth factor (bFGF; 0.6 ng/ml), endothelin-1 (endo-1; 10 nM), and alpha-melanocyte stimulating hormone (alpha-MSH; 10 nM). The latter two factors replaced the commonly used mitogens 12-O-tetradecanoylphorbol 13-acetate (TPA) and bovine pituitary extract (BPE), respectively. Basic FGF alone maintained the viability but did not induce the proliferation of melanocytes. The addition of endo-1 to the bFGF-containing medium resulted in reduction of tyrosinase activity without enhancement of proliferation. However, the addition of alpha-MSH to the bFGF-containing medium potentiated melanocyte proliferation and tyrosinase activity. The concomitant addition of endo-1, alpha-MSH, and bFGF significantly increased the entry of melanocytes into S phase and potentiated their proliferation. Melanocytes maintained under these conditions had the same tyrosinase activity as those maintained in a medium containing alpha-MSH and bFGF. The signal transduction pathway induced by either endo-1 or bFGF, but not alpha-MSH, includes the activation of the mitogen-activated (MAP) kinase pathway. The addition of both endo-1 and bFGF had more than an additive effect on the MAP kinase extracellular signal-regulated kinase 2 (ERK2). This effect was further increased by the addition of alpha-MSH to these two growth factors. In summary, we have devised a growth medium for human melanocytes based on the use of physiologic mitogens that substituted for routinely used artificial and undefined growth factors. The resulting cultures should be desirable for clinical uses and permissive for the expression of in vivo relevant responses to regulatory factors.

Adult↗
PubMed1995

Thy-1+ dendritic cells express truncated form of POMC mRNA.

The present study was designed to examine the expression of proopiomelanocortin (POMC) and its related derivative peptide adrenocorticotropic hormone (ACTH) in murine derived Thy-1+ dendritic cells. Immunostaining using a polyclonal antibody specific to ACTH and parent POMC molecule indicated the presence of POMC and its derivative peptide, ACTH, in cultures of Thy-1+ dendritic cells. To explore whether the POMC peptide is present as a reservoir or synthesized de novo in Thy-1+ dendritic cells. Northern blot analysis using 30-mer oligonucleotide probe for alpha-MSH/ACTH precursor POMC was carried out in total RNA from these cells. Northern blot analysis revealed the presence of POMC like mRNA transcript. However, the observed size of transcript was smaller (approximately 0.9 kb) than that expressed by murine AtT20 cells (approximately 1.2 kb), an anterior pituitary tumor cell line used as a positive control. These observations suggest that the epidermal Thy-1+ lymphocytes, like thymic lymphocytes, might serve the epidermis as one source for the synthesis of POMC. The synthesis and presence of POMC in the epidermis may be related to some of the pigmentary anomalies observed in many mucocutaneous disorders.

Adrenocorticotropic Hormone↗
PubMed1994

Terminal differentiation and senescence in the human melanocyte: repression of tyrosine-phosphorylation of the extracellular signal-regulated kinase 2 selectively defines the two phenotypes.

Melanocytes are pigmented cells distributed in humans in several organs like the epidermis, the leptomeninges, the eye, and the inner ear. Epidermal melanocytes, whether derived from adult or neonatal skin, proliferate well in a medium supplemented with phorbol esters and other mitogens before they undergo senescence. Potent cAMP inducers like cholera toxin are also growth promoters for neonatal melanocytes but only transient growth stimulators for cells derived from adults. We used this cellular system to delineate biochemical pathways involved in proliferation and in terminal differentiation. Here we show that after a period of 4-8 wk of sustained proliferation in the presence of cholera toxin, the adult melanocytes became round, flat, and enlarged. These changes were associated with terminal growth and preceded by a five- to sixfold increase in cAMP levels and an 8- to 10-fold increase in melanin content. The simultaneous addition of phorbol esters and cholera toxin did not prevent cells from reaching terminal differentiation. Identified targets for phorbol esters are protein kinase C (PKC) and the mitogen-activated kinases (MAPKs), also called extracellular signal-regulated kinases (ERKs). PKC was found to be similarly regulated in proliferating and in terminally differentiated melanocytes. Proliferating melanocytes in early or late passage showed identical activation of the kinase ERK2. This kinase was rapidly phosphorylated upon phorbol 12-myristate 13-acetate (PMA) addition and specifically accumulated in the nucleus of the cells, whereas in unstimulated cells it had a perinuclear distribution. In contrast, senescent and terminally differentiated cells were unable to phosphorylate tyrosine residues of the ERK2 gene product in spite of presenting normal amounts of ERK2 protein. In addition, ERK2 did not show the nuclear accumulation observed in proliferating melanocytes after PMA activation and remained localized in the perinuclear area. These results demonstrate that senescent and terminally differentiated melanocytes share a common block in a critical pathway thought to integrate multiple intracellular signals transmitted by various second messengers and specifically prevent the continuation of the signal transduction cascade initiated by PMA activation of PKC.

Adult↗
PubMed1994

Proliferation and propagation of human melanocytes in vitro are affected by donor age and anatomical site.

We report the effects of two factors, donor age and anatomical site, on the proliferation and melanization of human melanocytes (MC) derived from (1) neonatal foreskins, (2) adult foreskins, or (3) adult breast or arm skin. Two different growth media have been used for this purpose. Medium I supports the long-term proliferation of neonatal MC, and medium II supports the growth of both neonatal and adult MC. We found that neonatal foreskin MC proliferated equally well in both media for more than 12 passages. However, adult MC behaved differently in the two growth media. In very early passages (passages 1-5), all three types of MC proliferated well and reached comparable final cell densities in medium I, but adult foreskin MC proliferated at a higher rate than neonatal or adult non-foreskin MC in medium II. The non-foreskin adult MC had the least proliferative rate in medium II. Unlike neonatal MC, both adult foreskin and non-foreskin MC underwent a drastic reduction in their proliferative rate after only a few (9-10) passages in culture. While the three types of MC differed in their proliferative rates, they responded similarly to melanogenic stimulation by cholera toxin (CT) and isobutyl methylxanthine (IBMX). These results demonstrate that the proliferation of human MC in standard culture media is affected by the donor age and the anatomical site from which these cells are derived. We conclude that human MC are heterogeneous, and caution must be used in extrapolating the results of studies on MC derived from different anatomical sites or age groups.

Adult↗
PubMed1993

Chronic growth stimulation of human adult melanocytes by inflammatory mediators in vitro: implications for nevus formation and initial steps in melanocyte oncogenesis.

In the human epidermis, melanocytes are distributed at a distance from each other. In contrast, melanocytes in nevi, which are considered benign neoplasms of melanocytes, are grouped in nests. Although still not well defined, environmental factors are thought to play an important role in the development of nevi. We found that chronic growth stimulation by leukotriene C4, a compound found in increased amounts in inflamed skin, induced pleiotropic modifications in the normal melanocyte phenotype. These changes include loss of contact inhibition and formation of structures resembling tumor spheroids. In parallel with these changes, there was a constitutive expression of Fos protein. Switching these cultures to medium supplemented with phorbol ester sustained growth with reversion of the altered phenotype. In contrast, a cAMP stimulator, cholera toxin, induced features of terminal differentiation. Our findings suggest a role for inflammatory mediators in human epidermal melanocytes. This observation provides insight into melanocyte growth alterations which may have relevance in early stages of melanocyte oncogenesis.

Adult↗
PubMed1993

The increase in cytosolic levels of protein kinase C precedes the growth factor requirements during the transition stem cells to differentiated progeny in the MCF-7 breast tumor cells.

The expression of regulatory genes within the context of a differentiation program can have profound long-term consequences in tissues with permanent renewing populations. The breast tumor cell line MCF-7 retains in culture some of the characteristics of a unidirectional differentiation pathway. We show that the cytosolic activity of the regulatory enzyme protein kinase C (PKC) precedes and continues the sequence of maturation in pre-differentiated subpopulations derived from a stem cell fraction. However, the activity declines in the most differentiated, postmitotic fraction. These results indicate that PKC may be considered among the regulatory genes in MCF-7 cells that specify maturation of the stem cell progeny.

Breast Neoplasms↗
PubMed1993

Pigmentation and inhibition of wound contraction by cultured skin substitutes with adult melanocytes after transplantation to athymic mice.

Wound closure with cultured skin substitutes results in epithelium that is consistently hypopigmented. Hypothetically, addition of human melanocytes to cultured skin grafts may result in normal pigmentation of healed skin. Skin substitutes were composed of human epidermal keratinocytes and melanocytes, dermal fibroblasts, and collagen-glycosaminoglycan substrates, and were incubated for 12 d in media for keratinocyte growth (KG, n = 4), for keratinocyte differentiation containing four fatty acids and vitamin E with basic fibroblast growth factor (KDF, n = 6) or epidermal growth factor (KDE, n = 6), or for melanocyte growth (MG, n = 6) with phorbol ester and 5% fetal bovine serum. Skin substitutes were grafted orthotopically to full-thickness skin wounds (2 x 2 cm) on athymic mice, and scored for percent original wound size (+/- SEM), visible pigmentation (number pigmented/n), and positive staining for human leukocyte antigens (HLA)-ABC after 6 weeks on the mice. The data show that cultured skin grafts containing human melanocytes that are incubated in KDE or MG media have statistically significant reduction in wound contraction, 1:1 correlation of expression of pigment and HLA-ABC, and increased frequency of pigmentation after healing compared to incubation in KG or KDF media. Transmission electron microscopy confirmed the presence of melanocytes, melanosomes, and pigment transfer to keratinocytes in pigmented skin. These results suggest that survival and differentiated function of cultured epithelium can support melanization of skin, and that skin analogues exposed to phorbol ester in vitro can support skin pigmentation after wound healing.

Adult↗
PubMed1991

Metabolism of benzo[a]pyrene by human melanocytes in culture.

Benzo[a]pyrene (B[a]P) is a ubiquitous environmental pollutant and known skin carcinogen. In the present study, in vitro addition of [3H]B[a]P to normal human melanocytes in culture, isolated from adult human skin, resulted in the metabolism of [3H]B[a]P both intracellularly and extracellularly. HPLC analysis showed that [3H]B[a]P-9,10- and 7,8-diol were the major intracellular and extracellular metabolites followed by 3,6-quinone, 9-hydroxy and 3-hydroxy metabolites. Significant amounts of the [3H]B[a]P metabolites were found to be present in the sonicated cell suspension and culture medium as the glucuronide and sulfate conjugates. In total 37.3% of the [3H]B[a]P added in the culture medium was metabolized by melanocytes, of which 21.1% was quantified as the intracellular and 16.2% as the extracellular metabolites. Our data show that human melanocytes are capable of metabolizing B[a]P.

Benzo(a)pyrene↗
PubMed1991

Structural aberration of the rough endoplasmic reticulum and melanosome compartmentalization in long-term cultures of melanocytes from vitiligo patients.

Long-term cultures of melanocytes were established from 14 subjects with vitiligo and from five normal controls and analyzed ultrastructurally. Cultured melanocytes from 78.6% of the vitiligo patients demonstrated abnormalities that consisted of 1) dilation of the rough endoplasmic reticulum (RER), 2) circular RER profiles, and/or 3) membrane bound compartments of melanosomes. Cultured melanocytes from control subjects were predominantly normal with only one of the normal cultures demonstrating minimal circular RER profiles. The three unique abnormal structures in cultured vitiligo melanocytes were not always concomitantly expressed and could not be associated with any specific clinical feature of vitiligo. Quantitative analysis of the RER demonstrated that the profiles of dilated RER in cultured vitiligo melanocytes expressed a significant 1.5-2.8-times increase in mean cisternal area over cultured control melanocytes (i.e., 5.41-9.92 microns 2 versus 3.53 microns 2, respectively). The cisterna of the dilated RER profiles frequently contained floccular material that appeared to originate from the ribosomes, an indication that the floccular material may be translation products. The dilation of RER in melanocytes from the same patient persisted through repeated subculturing for up to 14.75 months. Epidermal melanocytes in biopsied skin from a patient whose cultured melanocytes were aberrant also demonstrated dilated and circular RER profiles. These results demonstrate that melanocytes from most vitiligo patients express an innate defect when cultured. Although this defect does not appear to be cytotoxic in vitro, this abnormality may be the primary defect that elicits melanocyte destruction in vivo.

Adolescent↗
PubMed1990

Adriamycin effects on hydroperoxide metabolism and growth of human breast tumor cells.

Human breast tumor cells MCF-7 were grown during 5 days in the presence of Adriamycin and the IC50 was 50 nM with the highest sublethal concentration 0.1 microM. At this latter concentration Adriamycin produced a complete inhibition of cell division and a partial reversion to a normal breast epithelial appearance. Similar effects of Adriamycin were observed in cells cultured in the presence of 10% FBS and in a chemically defined medium, with Se-glutathione peroxidase activities of 3.8 and 1.3 U/mg of protein, respectively. Cell size and cell oxygen uptake were increased by 41% and by 50%, respectively, in Adriamycin-treated cells. The spontaneous chemiluminescence of monolayers of intact MCF-7 cells (81 +/- 9 cps/mg protein) was increased by 48% in the Adriamycin-treated cultures (120 +/- 11 cps/mg of protein) in agreement with a 91% higher concentration of malondialdehyde in the same cultures. Adriamycin treatment produced a 71% increase in the steady state concentration of H2O2, which was estimated assuming diffusion equilibrium with the external medium, from 1.38 microM in the control cells to 2.38 microM in the treated cells. Cyanide-insensitive respiration was also higher in the cells exposed to the drug than in the control cells. Adriamycin did not affect the activity of the antioxidant enzymes, Cu-Zn and Mn-superoxide dismutase, Se and non-Se-glutathione peroxidase, and catalase. These results contribute to the current hypothesis that oxygen free radicals produced by Adriamycin redox cycling are responsible for at least part of the cytotoxic effects due to this drug.

Analysis of Variance↗
PubMed1990

Increased secretory activity and estradiol receptor expression are among other relevant aspects of MCF-7 human breast tumor cell growth which are expressed only in the absence of serum.

We compared the morphology, clonogenic ability, Percoll gradient distribution, estrogen receptor proteins, and interactions with mesenchymal cells in MCF-7 breast tumor cells grown in medium containing fetal calf serum and insulin (FCS-I) or in a defined medium with insulin (ID) as the only growth factor. In the absence of serum and at densities below 5000-8000 cells/cm2, MCF-7 cells required epidermal growth factor, insulin, and thrombin. When cells reached a density of 23,000-26,000 cells/cm2, only insulin was necessary for optimal growth. In ID medium cells showed an enlarged Golgi apparatus and marked plasma membrane modifications, suggesting increased secretory activity. Moreover there was an increase in the release of protein products to the culture medium and a time-dependent ability of these cells to form macrocolonies in soft agar. On the contrary, cells in FCS-I showed no Golgi complex and few plasma membrane modifications. In both culture media tight junctions, desmosomes, and tonofilaments were present. We investigated the effect of conditioned media from MCF-7 cells growing in FCS-I or ID on the growth of primary rat vaginal fibroblasts. The growth of these mesenchymal cells was stimulated by FCS-I medium and inhibited by ID medium. By contrast, the embryonic fibroblast (preadipocyte) line CHEF/18 was also stimulated by FCS-I for the first 48 h, but thereafter ceased growth and acquired lipid droplets and a differentiated morphology. With ID medium, CHEF/18 cells were only partially inhibited with no changes in morphology. The Percoll gradient profiles of ID cells showed the same six fractions of increasing density as recently described. However, there was a progressive increase in subpopulations with higher growth rates and a decrease in the relative amount of the most differentiated cells. A unique feature of the growth analysis of MCF-7 cells in the absence of serum is the increased expression of the estradiol receptor gene. These studies show that the growth and differentiated properties of tumor cells can depend upon the cellular environment and offer a model system in which to further study this modulation.

Animals↗
PubMed1990

Successful culture of adult human melanocytes obtained from normal and vitiligo donors.

The development of growth conditions for human melanocytes from uninvolved skin from vitiligo patients and age-matched normal adults is a prerequisite to understanding the etiology of this pigmentary disorder. By using new growth conditions, pure melanocyte cultures were prepared from normal adults and the pigmented skin of vitiligo donors. Both cell types grew without a lag period and were maintained for more than six months (4-8 passages). The cultures could be expanded from several thousand melanocytes in the original cell suspension to several million cells (2-7 x 10(6] in pure culture. To obtain these results, the current standard conditions for the culture of fetal melanocytes were substantially modified. MEM-S medium was less satisfactory than MCDB-153. Adult normal and vitiligo cells also required the presence of exogenous catalase (20 micrograms/ml) during isolation and primary seeding. Thereafter, this enzyme was not necessary. Melanocytes grew best and gave the highest yields if the concentrations of both calcium (200 microM) and the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) were low (4-8 nM). Other growth factors included in the MCDB-153 media were bFGF, crude bovine pituitary extract, insulin, transferrin, hydrocortisone, 5% FCS, and the antioxidant alpha-tocopherol. Cholera toxin and isobutylmethylxanthine (IBMX) were omitted from the MCDB-153 growth medium because they slowed down growth even at very low concentrations. The results indicate adult and vitiligo melanocytes can be cultured. Preliminary studies of the normal and vitiligo cells indicate that vitiligo melanocytes retain some of the ultrastructural abnormalities observed in skin even when grown in culture.

1-Methyl-3-isobutylxanthine↗
PubMed1989

Growth factors and hormones which affect survival, growth, and differentiation of the MCF-7 stem cells and their descendants.

The human breast tumor cell line was separated by Percoll density gradient centrifugation into six different subpopulations, A to F, one of which (E) appears to contain the stem cells on the basis of several criteria (M. Resnicoff et al. 1987, Proc. Natl. Acad. Sci. USA 84, 7295. We now analyzed the response of the isolated subpopulations to insulin, thrombin, PGF2 alpha, estradiol, and 13-cis-retinal. We demonstrate that the first two growth factors stimulate [3H]thymidine incorporation in the more differentiated subpopulations (D and F), while PGF2 alpha has mitogenic activity in subpopulations C and D. In the absence of any added growth factor, estradiol has the extreme and transient capacity of allowing the stem cell to detach from the tissue culture dish and to grow in suspension as multicellular aggregates (MCF-7/SE cells). 13-cis-Retinal acts as a negative modulator of differentiation and protects the cells from the inhibitory and differentiation activity of Na-butyrate.

Breast Neoplasms↗
PubMed1988

Protein kinase C is differentially regulated by thrombin, insulin, and epidermal growth factor in human mammary tumor cells.

The exposure of serum-deprived mammary tumor cells MCF-7 and T-47D to insulin, thrombin, and epidermal growth factor (EGF) resulted in dramatic modifications in the activity and in the translocation capacity of protein kinase C from cytosol to membrane fractions. Insulin induces a 600% activation of the enzyme after 5 h of exposure to the hormone in MCF-7 cells; thrombin either activates (200% in MCF-7) or down-regulates (in T-47D), and EGF exerts only a moderate effect. Thus, the growth factors studied modulate differentially the protein kinase C activity in human mammary tumor cells. The physiological significance of the results obtained are discussed in terms of the growth response elicited by insulin, thrombin, and EGF.

Breast Neoplasms↗
PubMed1988

Effect of estradiol and tamoxifen on the anchorage-independent growth of the subpopulations derived from MCF-7 breast carcinoma cells: cytogenetic analysis of the stem cell subpopulation.

The MCF-7 breast carcinoma cell line can be separated by Percoll density gradient centrifugation into several subpopulations, A to F, one of which (E) has been previously suggested to be highly enriched in stem cells. The anchorage-independent growth of the different fractions and its sensitivity to estradiol (E2) and tamoxifen (TAM) was assayed. The anchorage-independent growth capacity of the different fractions was E greater than A greater than B greater than D greater than C,F. The E fraction had the highest clonogenic index (6.62 +/- 1.18) and was unaffected by E2 or TAM. The karyotypic analysis of the E fraction revealed features similar to those of the unfractionated cell line. It is suggested that the high growth rate of fraction E is due to an enrichment in stem cells and not to the existence of a different clone.

Breast Neoplasms↗
PubMed1987

13-cis-retinal stimulates proliferation and induces intranuclear protein accumulation in the human mammary tumor cells MCF-7.

The human mammary tumor cells MCF-7 show enhanced proliferation when treated with low doses (10(-8)-10(-7) M) of 13-cis Retinal (a vitamin A derivative). These results are independent of the growth medium used. We describe a novel effect of 13-cis Retinal: the increased synthesis and accumulation of nuclear proteins in chronically treated cells. The cytoplasmic proteins and proteins released to the culture medium are transiently and oppositly modified. Moreover, chronic treated cells have growth advantages over the untreated counterparts in a clonogenic soft agar assay.

Breast Neoplasms↗