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Biomedical subjects

Barbara A Gilchrest

Publications and source records attributed to Barbara A Gilchrest.

At least 37 records · Page 2Linked to original sources
PubMed2005

Modulation of vascular endothelial growth factor receptors in melanocytes.

Vascular endothelial growth factor (VEGF) is constitutively produced by keratinocytes, but has no known epidermal target cell. We now report that normal human melanocytes (Mc) maintained in serum-free, hormone-, and growth factor-supplemented medium lacking phorbol ester and choleragen constitutively express VEGF receptor-1 (VEGFR-1), VEGFR-2, and neuropilin-1. Furthermore, stimulation of Mc with VEGF165 isoform leads to phosphorylation of VEGFR-2, the receptor responsible for most of the VEGF-mediated effects in endothelial cells, suggesting that the receptor is functional. Interestingly, in Mc, VEGFR-2 expression is induced by ultraviolet irradiation and is downregulated by VEGF and tumor necrosis factor-alpha. Prolonged culture (>8 weeks) in the presence of phorbol ester abrogates VEGFR-2 expression, explaining previous reports that Mc do not express VEGFR-1 and VEGFR-2. These data suggest that VEGF may play a role in Mc behavior in skin.

Blotting, Western↗
PubMed2005

The tale of the telomere: implications for prevention and treatment of skin cancers.

Work in many laboratories over the past decade has established a central role for the telomere in maintaining genomic integrity. Available data may be interpreted to indicate that telomere disruption, whether due to acute DNA damage or progressive telomere shortening, is the initial event that triggers multiple DNA damage responses. The specific initiating event is likely exposure of the otherwise concealed single-stranded 3' overhang, tandem repeats of TTAGGG, a signal that can be provided to cells in the absence of DNA damage by exogenously provided T-oligos. The ability of T-oligo treatment to trigger SOS-like responses and/or to cause selective apoptosis of already malignantly transformed cells may provide an important new means of cancer prevention and treatment.

Animals↗
PubMed2004

Bone morphogenetic protein (BMP) signaling controls hair pigmentation by means of cross-talk with the melanocortin receptor-1 pathway.

Hair pigmentation is controlled by tightly coordinated programs of melanin synthesis and involves signaling through the melanocortin type 1 receptor (MC-1R) that regulates the switch between pheomelanogenesis and eumelanogenesis. However, the involvement of other signaling systems, including the bone morphogenetic protein (BMP) pathway, in the control of hair pigmentation remains to be elucidated. To assess the effects of BMP signaling on hair pigmentation, transgenic mice overexpressing the BMP antagonist noggin (promoter: keratin 5) were generated. Whereas wild-type C3H/HeJ mice have a subapical yellow band on otherwise black dorsal hairs, K5-Noggin mice are characterized by the absence of a yellow band and near-black pigment in dorsal coat. Noggin overexpression is accompanied by strongly reduced levels of Agouti signal protein and enhanced expression of microphthalmia transcription factor in the midphase of the hair-growth cycle. Wild-type color in K5-Noggin mice is restored by administration of a synthetic MC-1R antagonist resulting in the reappearance of a subapical yellow band. BMP-4 stimulates the expression of Agouti transcripts and protein in primary epidermal keratinocytes, and BMP signaling positively regulates dermal papilla-specific enhancer of the Agouti gene in primary dermal fibroblasts. Taken together, these data suggests that BMP signaling controls the expression of Agouti protein in the hair follicle and provide evidence for interaction between BMP and MC-1R signaling pathways to modulate the balance between pheomelanogenesis and eumelanogenesis during hair growth.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Signaling pathway requirements for induction of senescence by telomere homolog oligonucleotides.

Cellular senescence is a major defense against cancer. In human fibroblasts, suppressing both the p53 and pRb pathways is necessary to bypass replicative senescence as well as senescence induced by ectopic expression of a dominant negative form of the telomere repeat binding factor 2, TRF2(DN). We recently reported that exposure to oligonucleotides homologous to the telomere 3' overhang (T-oligos) activates both the p53 and pRb pathways and leads to senescence in primary human fibroblasts. To further characterize T-oligo-induced senescence, we compared established isogenic fibroblast cell lines lacking functional p53 and/or pRb pathways to the normal parental line. Here, we report that, as in physiologic senescence, inactivation of both the p53 and pRb pathways is necessary to suppress T-oligo-induced senescence. Moreover, T-oligo rapidly induces senescence in a malignant fibroblast-derived cell line, demonstrating the potential of using T-oligo as a novel anticancer therapeutic. Our data support the hypothesis that exposure of the TTAGGG tandem repeat telomere 3' overhang sequence is the event that initiates signaling through DNA damage response pathways after experimental telomere disruption, serial passage, or acute genomic damage of normal cells.

Base Sequence↗
PubMed2004

Fas signaling is involved in the control of hair follicle response to chemotherapy.

Chemotherapeutic agents induce p53-dependent apoptosis in the hair follicle (HF) resulting in hair loss, a common side effect of cancer therapy. Here, we show that Fas as a p53 target plays important role in the HF response to cyclophosphamide. Specifically, we demonstrate that Fas is up-regulated in HF keratinocytes after cyclophosphamide treatment, Fas ligand-neutralizing antibody partially inhibits HF response to cyclophosphamide in wild-type mice, and Fas knockout mice show significant retardation of cyclophosphamide-induced HF involution associated with reduced Fas-associated death domain and caspase-8 expression. These data raise a possibility to explore blockade of Fas signaling as a part of complex local therapy for inhibiting keratinocyte apoptosis and hair loss induced by chemotherapy.

Adaptor Proteins, Signal Transducing↗
PubMed2004

The receptor for activated C-kinase-I (RACK-I) anchors activated PKC-beta on melanosomes.

Protein kinase C (PKC), a family of at least eleven isoforms, mediates numerous cell functions. In human melanocytes, alpha, beta, delta, epsilon and zeta isoforms of PKC are expressed, but uniquely PKC-beta activates tyrosinase, the key and the rate-limiting enzyme in melanogenesis, by phosphorylating specific serine residues on its cytoplasmic domain. To investigate the mechanism by which only PKC-beta phosphorylates tyrosinase, we examined the expression of receptor for activated C-kinase-I (RACK-I), a receptor specific for activated PKC-beta, on the surface of melanosomes, the specialized organelle in which melanogenesis occurs. Immunoblot analysis of purified melanosomes revealed that RACK-I is readily detectable. Immunoprecipitation of RACK-I from purified melanosomes, followed by immunoblot analysis using antibody against PKC-beta, revealed abundant PKC-beta, whereas PKC-alpha was not detected when immunoblot analysis was performed using antibody against PKC-alpha. Activation of PKC in melanocytes increased the level of PKC-beta co-immunoprecipitated with RACK-I, while the level of melanosome-associated RACK-I decreased when melanocytes were treated chronically with the 12-0-tetradecanoyl-phorbol 13-Acetate (TPA), a condition known to deplete PKC and reduce tyrosinase activity. Immunoprecipitation with RACK-I antibody co-precipitated fewer PKC-beta in the presence of UV-activated 1, 1'-decamethylenebis-4-aminoquinaldinium di-iodide (DECA), known to disrupt the interaction between activated PKC-beta and RACK-I. Treatment of intact melanocytes with DECA also decreased tyrosinase activity. Moreover, suppression of RACK-I expression by transfecting melanocytes with siRNA against RACK-I reduced the basal tyrosinase activity and blocked TPA-induced increases in tyrosinase activity. Taken together, these results demonstrate that RACK-I anchors activated PKC-beta on the melanosome membrane, allowing PKC-beta to phosphorylate tyrosinase.

Base Sequence↗
PubMed2004

Topical DNA oligonucleotide therapy reduces UV-induced mutations and photocarcinogenesis in hairless mice.

UV-induced DNA damage gives rise to mutations and skin cancer. We have previously reported that treatment of skin cells in vitro with thymidine dinucleotide (pTT) activates p53 and increases the ability of cells to repair subsequent UV-induced DNA damage by enhancing endogenous DNA repair capacity. Here we show that topical pTT pretreatment enhances the rate of DNA photoproduct removal, decreases UV-induced mutations, and reduces photocarcinogenesis in UV-irradiated hairless WT repair-proficient and Xpc(+/-) heterozygous partially repair-deficient mice, both transgenic for the lacZ/pUR288 mutation-indicator gene. These data support the existence of inducible mammalian DNA damage responses that increase DNA repair capacity after DNA damage and hence reduce the impact of future exposures to environmental carcinogens. The ability of topically applied pTT to induce protective physiologic responses that normally result from DNA damage suggests a previously undescribed means of reducing skin cancer in high-risk individuals.

Administration, Topical↗
PubMed2004

A trial of short incubation, broad-area photodynamic therapy for facial actinic keratoses and diffuse photodamage.

BACKGROUND: There is no completely satisfactory treatment for multiple actinic keratoses (AKs). OBJECTIVE: To evaluate the efficacy of short incubation, broad-area application of delta-aminolevulinic acid followed by exposure to activating light-photodynamic therapy (delta-ALA/PDT) for treatment of AKs and background photodamage. The benefit of pretreatment with 40% urea cream to enhance penetration and the use of topical 3% lidocaine hydrochloride to decrease discomfort were also evaluated. METHODS: Eighteen patients with at least 4 nonhypertrophic facial AKs and mild to moderate diffuse facial photodamage were enrolled in the study. For 7 days, 40% urea cream or vehicle was applied to half of the treatment area, and then delta-ALA was applied to the entire area for 1, 2, or 3 hours. Lidocaine hydrochloride (3%) or vehicle cream was also applied to the entire area 45 minutes before exposure to 10 J/cm(2) of blue light. Pain,phototoxic reactions, AK counts, and photodamage improvement were evaluated 1 day, 1 week, and 1 month after treatment in all patients and after 5 months in 10 patients. RESULTS: All patients experienced mild to moderate discomfort during treatment and moderate phototoxic effects for 1 week. At 1 and 5 months there was significant reduction in AKs in all groups and significant improvement of several photodamage parameters. Different delta-ALA application times and pretreatment with urea cream or lidocaine had no significant effect on the results. CONCLUSIONS: This delta-ALA/PDT protocol is safe and effective for AK treatment as well as for improving photodamage. Further studies with a larger cohort, longer follow-up, and histologic confirmation of the clinical data would be of value.

Administration, Topical↗
PubMed2004

Topical application of a protein kinase C inhibitor reduces skin and hair pigmentation.

To determine whether inhibition of PKC-beta activity decreases pigmentation, paired cultures of primary human melanocytes were first pretreated with bisindolylmaleimide (Bis), a selective PKC inhibitor, or vehicle alone for 30 min, and then treated with TPA for an additional 90 min to activate PKC in the presence of Bis. Bis blocked the expected induction of tyrosinase activity by activation of PKC. Addition of a peptide corresponding to amino acids 501-511 of tyrosinase containing its PKC-beta phosphorylation site, a presumptive PKC-beta pseudosubstrate, gave similar results. To determine whether Bis reduces pigmentation in vivo, the backs of four shaved and depilated pigmented guinea pigs were UV irradiated with a solar simulator for 2 wk excluding weekends. Compared to vehicle alone, Bis (300 microM), applied twice daily to paired sites for various periods encompassing the irradiation period, decreased tanning. Bis also, although less strikingly, reduced basal epidermal melanin when topically applied twice daily, 5 d per wk, for 3 wk to shaved and depilated unirradiated skin. Moreover, topical application of Bis (100 microM) once daily for 9 d to the freshly depilated backs of 8-wk-old mice markedly lightened the color of regrowing hair. These results demonstrate that inhibiting PKC activity in vivo selectively blocks tanning and reduces basal pigmentation in the epidermis and in anagen hair shafts.

Animals↗
PubMed2004

The interdependence of skin aging, skin cancer, and DNA repair capacity: a novel perspective with therapeutic implications.

The human body is constantly exposed to exogenous and endogenous insults that threaten its genomic integrity and that lead to changes at the molecular, biochemical, and cellular levels. As a major interface between the environment and the internal milieu, our skin is especially subject to such events. Common insults include but are not limited to infectious agents, environmental pollutions and toxins, carcinogens, and ultraviolet (UV) irradiation. It is estimated that there are thousands of DNA alterations in each cell daily. Therefore, if not efficiently repaired, our genome would rapidly be destroyed. This review focuses predominantly on UV-induced DNA damage in human skin, protective molecular responses to UV damage, and the consequences of these opposing forces for aging and photocarcinogenesis.

DNA Damage↗
PubMed2004

Telomere-based DNA damage responses: a new approach to melanoma.

Melanoma is the most fatal skin cancer, often highly resistant to chemotherapy. Here we show that treatment with an 11-base DNA oligonucleotide homologous to the telomere 3' overhang sequence (T-oligo) induces apoptosis of several established human melanoma cell lines, including the aggressive MM-AN line, whereas normal human melanocytes exposed to the same or higher T-oligo concentrations show only transient cell cycle arrest, implying that malignant cells are more sensitive to T-oligo effects. When MM-AN cells were briefly exposed to T-oligo in culture and injected into the flank or tail vein of SCID mice, eventual tumor volume and number of metastases were reduced 85-95% compared with control mice. Similarly, T-oligos administered intralesionally or systemically selectively inhibited the growth of previously established MM-AN tumor nodules in the flank and peritoneal cavity by 85 to 90% without detectable toxicity. We previously showed that T-oligos act through ATM, p95/Nbs1, E2F1, p16INK4A, p53, and the p53 homologue p73 to modulate downstream effectors and now additionally demonstrate striking down-regulation of the inhibitor of apoptosis protein livin/ML-IAP. We suggest that T-oligo mimics a physiologic DNA damage signal that is frequently masked in malignant cells and thereby activates innate cancer prevention responses. T-oligos may provide a novel therapeutic approach to melanoma.

Animals↗
PubMed2004

Solar-simulated irradiation evokes a persistent and biphasic IL-1alpha response.

Exposure of skin to solar-simulated irradiation generates a multitude of adaptive responses including cytokine transcription, synthesis and secretion. Interleukin-1 (IL-1) is one of the cytokines induced in epidermal cells in response to UV irradiation. It displays a broad range of mitogenic and inflammatory activities including fibroblast proliferation and T-cell activation. There are two forms, IL-1alpha and IL-1beta; and IL-1alpha is the predominant form secreted by epidermal keratinocytes. UV-induced modulations of IL-1alpha message levels have been extensively studied within the first 48 h after irradiation, but longer term changes and impact on IL-1alpha cellular protein levels are virtually unexplored. We now report that cells of keratinocyte origin (SCC 12F) respond to a single physiologic dose of solar-simulated irradiation with both early (8 h) and late (72 h) peaks of IL-1alpha mRNA induction. UV-stimulated IL-1alpha secretion is increased above sham-irradiated control secretion for at least 96 h after irradiation. Our study provides evidence that UV-induced adaptive cutaneous responses persist for at least several days, and suggests that different mechanisms may mediate the early vs. late inductions.

Carcinoma, Squamous Cell↗
PubMed2004

delta-Aminolevulinic acid and blue light photodynamic therapy for treatment of multiple basal cell carcinomas in two patients with nevoid basal cell carcinoma syndrome.

BACKGROUND: Patients with nevoid basal cell carcinoma syndrome suffer from multiple basal cell carcinomas, requiring numerous surgical procedures that over time leave them with multiple disfiguring scars. Photodynamic therapy with delta-aminolevulinic acid using red light (approximately 630 nm) sources has been reported as effective in treatment of superficial and small nodular basal cell carcinomas. To our knowledge, the blue light source (417 nm peak irradiance) approved by the FDA for treatment of actinic keratoses has not been used for photodynamic therapy with delta-aminolevulinic acid of basal cell carcinoma. OBJECTIVE: We report treatment of two nevoid basal cell carcinoma syndrome patients, women aged 21 and 47, with 20%delta-aminolevulinic acid solution and 417-nm blue light source (irradiance 10 mW/cm(2)). METHODS: delta-Aminolevulinic acid was applied topically on lesions 1 to 5 hr before light treatment. Lesions were illuminated with 417+/-5-nm blue light for 1000 sec (10 J/cm(2)). Two consecutive treatments 1 week apart were administered as a therapeutic course. Each patient underwent two courses of photodynamic therapy with delta-aminolevulinic acid 2 to 4 months apart. The reported assessment was made 8 months after initial treatment. In most sessions the entire face, rather than visible basal cell carcinomas only, was treated. The treated basal cell carcinomas were clinically subdivided to superficial or nodular type guided by their morphologic features. A total of 9 superficial and 16 nodular basal cell carcinomas on the face and 27 superficial basal cell carcinomas on the lower extremities were treated. RESULTS: Complete clinical response was observed in 8 of 9 (89%) superficial basal cell carcinomas and 5 of 16 (31%) nodular basal cell carcinomas on the face and in 18 of 27 (67%) of superficial basal cell carcinomas on the lower extremities. The remaining 21 lesions showed partial clinical resolution. No new basal cell carcinomas were observed during the 8-month follow-up period in areas treated with a broad application technique. Resolution of the lesions was accompanied by an excellent cosmetic outcome and decreased prominence of old surgical scars in the more severely affected patient. Treatments were well tolerated, but associated with moderate to severe stinging during illumination. CONCLUSION: To our knowledge this is the first use of photodynamic therapy with delta-aminolevulinic acid with 417-nm blue light for treatment of multiple basal cell carcinomas in patients with nevoid basal cell carcinoma syndrome. Our clinical results demonstrate that the blue light reduces cutaneous tumor burden in such patients. Further studies are needed to confirm that broad-area photodynamic therapy with delta-aminolevulinic acid may eradicate subclinical tumors in nevoid basal cell carcinoma syndrome sufferers, as suggested by a strikingly decreased incidence of new basal cell carcinomas in our patients.

Adult↗
PubMed2003

Noggin overexpression inhibits eyelid opening by altering epidermal apoptosis and differentiation.

Contact of developing sensory organs with the external environment is established via the formation of openings in the skin. During eye development, eyelids first grow, fuse and finally reopen, thus providing access for visual information to the retina. Here, we show that eyelid opening is strongly inhibited in transgenic mice overexpressing the bone morphogenetic protein (BMP) antagonist noggin from the keratin 5 (K5) promoter in the epidermis. In wild-type mice, enhanced expression of the kinase-inactive form of BMPR-IB mediated by an adenovirus vector also inhibits eyelid opening. Noggin overexpression leads to reduction of apoptosis and retardation of cell differentiation in the eyelid epithelium, which is associated with downregulation of expression of the apoptotic receptors (Fas, p55 kDa TNFR), Id3 protein and keratinocyte differentiation markers (loricrin, involucrin). BMP-4, but not EGF or TGF-alpha, accelerates opening of the eyelid explants isolated from K5-Noggin transgenic mice when cultured ex vivo. These data suggest that the BMP signaling pathway plays an important role in regulation of genetic programs of eyelid opening and skin remodeling during the final steps of eye morphogenesis.

Adenoviridae↗
PubMed2003

UV induces VEGF through a TNF-alpha independent pathway.

Vascular endothelial growth factor (VEGF) is a potent keratinocyte-derived angiogenic factor. Prior reports suggest that following UV irradiation VEGF in keratinocytes is induced primarily by tumor necrosis factor (TNF)- alpha, a cytokine synthesized and secreted by keratinocytes after UV irradiation. We investigated whether blocking TNF-alpha binding to its receptors would inhibit UV-induced VEGF expression and secretion in the keratinocyte-derived line SCC-12F. Irradiation with physiologic UV doses (30 mJ/cm2) substantially induced VEGF mRNA in this cell line, as expected, and mRNA induction was followed by increased VEGF in medium conditioned by UV-irradiated cells. Also as expected, TNF-alpha induced VEGF expression and secretion in a dose-dependent manner. Addition of a hexapeptide (Ac-KWIIVW-NH2), known to block TNF-alpha binding to its receptors, abrogated this TNF-alpha effect on VEGF mRNA induction. However, addition of the peptide to cells immediately after UV irradiation did not substantially affect VEGF mRNA induction or secretion into the medium. Our results suggest that VEGF induction after UV irradiation is mediated by multiple mechanisms and that blocking a single pathway does not affect the response.

Animals↗