PubMed Health⌕ Search

Biomedical subjects

D R Roop

Publications and source records attributed to D R Roop.

At least 73 records · Page 4Linked to original sources
PubMed1995

Mutations in the 1A domain of keratin 9 in patients with epidermolytic palmoplantar keratoderma.

Epidermolytic palmoplantar keratoderma is an autosomal dominant skin disorder characterized by hyperkeratosis of the palms and soles. Ultrastructurally the disease exhibits abnormal keratin filament networks and tonofilament clumping like that found in the keratin disorders of epidermolysis bullosa simplex and epidermolytic hyperkeratosis. The disease has been mapped to chromosome 17q11-q23 in the region of the type 1 keratin gene locus and more recently mutations have been found in the palmoplantar specific keratin, keratin 9. We have analyzed six unrelated incidences of epidermolytic palmoplantar keratoderma for mutations in their keratin 9 genes. In two of these, we have identified mutations that alter critical residues within the highly conserved helix initiation motif at the beginning of the rod domain of keratin 9. In a three-generation Middle Eastern kindred we found a C to T transition at codon 162 that results in an arginine to tryptophan substitution at position 10 of the 1A alpha-helical domain, thus confirming this codon as a hot spot for mutation in keratin 9. The other mutation found involves a T to C transition at codon 167 that results in the expression of a serine residue in place of the normal leucine at position 15 of the 1A segment and is the first documentation of this mutation in this gene. The identification of these substitutions extends the current catalog of disease causing mutations in keratin 9.

Base Sequence↗
PubMed1995

12-O-tetradecanoylphorbol-13-acetate promotion of transgenic mice expressing epidermal-targeted v-fos induces rasHA-activated papillomas and carcinomas without p53 mutation: association of v-fos expression with promotion and tumor autonomy.

Transgenic mice that expressed v-fos exclusively in the epidermis by means of a human keratin K1-based targeting vector (HK1.fos) developed preneoplastic epidermal hyperplasia and hyperkeratosis after long latency and an associated wound promotion stimulus. To assess the requirements for papilloma formation and malignant conversion and determine the sensitivity to a chemical promotion stimulus, HK1.fos mice were promoted with 12-O-tetradecanoylphorbol-13-acetate (TPA). HK1.fos mice were sensitive to TPA promotion but developed papillomas only after long latency (20-30 weeks of promotion) and in relatively few numbers per animal, suggesting the necessity of an additional genetic event prior to overt lesion formation. Consistent with this idea, at 60 weeks, on cessation of TPA promotion, these HK1.fos TPA-papillomas were found to be autonomous, TPA-independent tumors which persisted, grew larger, and converted to malignancy. Analysis of HK1.fos tumor RNA and DNA identified endogenous c-rasHa mutations at codons 12 and 61 in papillomas and carcinomas; however, no p53 tumor suppressor gene mutations were detected. These data indicate that epidermal expression of v-fos induces sensitivity to TPA promotion, but since additional genetic events, such as endogenous c-rasHa activation, appear to be required in tumorigenesis, v-fos may predominantly play a role in the mechanism of promotion to achieve papilloma autonomy and TPA independence. Furthermore, spontaneous malignant conversion in this model does not appear to involve mutations in the p53 tumor suppressor gene.

Animals↗
PubMed1995

Differentiation and tumor response to retinobenzoic acid RE-80 in a malignant conversion model.

The synthetic retinobenzoic acid RE-80 was evaluated for its potential as an inductor of tumor cell differentiation and as a chemopreventive agent. A minimally toxic dose of RE-80 in vitro produced morphologic changes typical differentiation in epidermal tumor cell colonies. Indirect immunofluorescence indicated induction of a differentiation-associated keratin of internal stratified epithelia, K13, and inhibition of the differentiation-associated epidermal keratin K1. Cultured cells were skin-grafted to athymic nu/nu mice to evaluate RE-80 effects on early stage malignant progression in vivo. When tumors had grown to 3 to 4 mm in diameter, mice were treated by intraperitoneal injection with RE-80 (67 micrograms, 170 mmol, i.p., two times per week) or vehicle (100 microliters 20% ethanol). Papillomas (benign) and moderately differentiated squamous cell carcinomas were reduced in volume about 4-fold by RE-80 treatment. Larger, poorly differentiated squamous cell carcinomas were unaffected. RE-80 resulted in a lower proportion of proliferative cells (detectable by bromodeoxyuridine incorporation) and a higher proportion of moderately to well differentiated tumors after 40 days of treatment compared with control tumors, which were mainly poorly differentiated squamous cell carcinomas. K13 induction in vitro was correlated with response to retinoid in vivo. Induction of differentiation may be mechanism of the response to RE-80 in vivo since carcinoma cells expressing K13 did not incorporate bromodeoxyuridine and were on a terminal pathway.

Animals↗
PubMed1994

Characterization of the mouse loricrin gene: linkage with profilaggrin and the flaky tail and soft coat mutant loci on chromosome 3.

Loricrin is the major component of a specialized structure, termed the cornified cell envelope, that is formed beneath the plasma membrane of stratified squamous epithelial cells and is coexpressed with profilaggrin in terminally differentiating epidermal keratinocytes. Full-length cDNAs for both mouse and human loricrin have been cloned and characterized, as has the human gene. Here we report the isolation and characterization of the mouse loricrin gene. The gene has a simple structure consisting of a single intron of 1091 bp within the 5' noncoding sequence and an uninterrupted open reading frame. Using PCR analyses of DNAs isolated from mouse x Chinese hamster somatic cell hybrids, we have mapped both the loricrin and the profilaggrin genes to chromosome 3. Genetic linkage analysis has shown that mouse loricin and profilaggrin lie within 1.5 +/- 1.1 centimorgans of each other. We have further shown that both genes map in the vicinity of the flaky tail (ft) and soft coat (soc) loci. These mouse mutants exhibit a number of changes in their integument, suggesting that abnormalities in these genes may contribute to the mutant phenotype.

Amino Acid Sequence↗
PubMed1994

Differentiation-specific expression of human keratin 1 is mediated by a composite AP-1/steroid hormone element.

Human keratin 1 (HK1) expression is associated with the loss of mitotic activity in epidermal keratinocytes and restricted to an intermediate stage of terminal differentiation. Recently, the control elements that mediate this differentiation-specific expression were identified (Huff, C. A., Yuspa, S. H., and Rosenthal, D. (1993) J. Biol. Chem. 268, 377-384). We now report the characterization of one of these elements. Footprint analysis on a 249-base pair fragment containing the calcium responsive element (CaRE) revealed two adjacent protected regions. The 5' most footprint contains an AP-1 consensus sequence while the 3' footprint encodes two inverted repeats of the canonical hormone response recognition sequence. Deletion of the AP-1-protected region abolished the calcium response in a reporter construct. Calcium activation of the reporter construct containing the intact CaRE was unaffected by the addition of thyroid hormone or estrogen. However, vitamin D3 was able to suppress calcium induction by the CaRE, and this suppression could be abrogated by the coaddition of retinoic acid. These studies show that AP-1 factors bind to the 5' element to mediate the calcium response while members of the steroid hormone receptor superfamily interact with the 3' element to modulate the calcium response.

Base Sequence↗
PubMed1994

Ornithine decarboxylase expression in cutaneous papillomas in SENCAR mice is associated with altered expression of keratins 1 and 10.

The patterns of expression of ornithine decarboxylase (ODC) and a number of keratinocyte differentiation products were examined in early papillomas by immunocytochemistry as an initial effort to develop phenotypic markers of the early stages of mouse skin tumorigenesis. Tumors were induced in SENCAR mice by initiation with 7,12-dimethylbenzanthracene, followed by once or twice weekly promotion treatments with 12-O-tetradecanoylphorbol-13-acetate. The patterns of immunocytochemical staining observed in early papillomas, collected after 7 weeks of promotion, were compared to those obtained with control skin and large, hyperkeratotic papillomas, collected after 23 weeks of promotion. In groups receiving 12-O-tetradecanoylphorbol-13-acetate, constitutive and induced ODC expression were evaluated either several days or 4.5 h after the last treatment, respectively. The major differentiation products in suprabasal keratinocytes are keratins, K1 and K10. These keratins were normally expressed in mildly hyperplastic epidermis, but staining became patchy in markedly hyperplastic epidermis. In early papillomas, K1 staining was patchy, and K10 staining occurred in very limited focal areas or was negative, such that the absence of staining delineated the lesions. Antibodies to the basal cell keratins, K5 and K14, stained both basal and suprabasal cells in hyperplastic epidermis and papillomas. Similarly, an antibody to keratin K6, which is expressed under conditions of hyperproliferation, uniformly stained the basal and suprabasal layers of hyperplastic epidermis and papillomas, demonstrating that the staining patterns observed with the antibodies to K1 and K10 were specific. Expression of ODC in the histologically normal skin of control mice was detected only in the hair follicles and depended on the hair cycle. Staining for induced ODC in mice treated with 12-O-tetradecanoylphorbol-13-acetate once weekly for 7 weeks was intense and diffuse throughout the suprabasal layers of the epidermis. In early and large papillomas, staining for constitutively expressed ODC was intense and diffuse in suprabasal cells. This intense staining for ODC occurred consistently in areas with decreased K1 and K10 expression, and, therefore, was associated with an altered pattern of differentiation. High constitutive ODC expression and decreased K1 and K10 expression will be useful phenotypic markers for studying the early stages of tumorigenesis in mouse skin.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1994

Epidermal expression of transforming growth factor-alpha in transgenic mice: induction of spontaneous and 12-O-tetradecanoylphorbol-13-acetate-induced papillomas via a mechanism independent of Ha-ras activation or overexpression.

To assess the requirements for papilloma formation in transgenic mice that overexpress transforming growth factor-alpha (TGF-alpha) in the epidermis (HK1.TGF alpha), we tested the sensitivity of HK1.TGF alpha mice to tumor promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA) and analyzed the resultant papillomas for synergic c-Ha-ras activation and overexpression. We observed that HK1.TGF alpha mice were highly sensitive to TPA promotion, exhibiting multiple papillomas as early as the third week of treatment. After 60 wk of promotion, malignant conversion was not observed and tumors regressed upon removal of the TPA promotion stimulus. Most of the TPA-induced papillomas did not have detectable c-Ha-ras mutations at codons 12, 13, or 61, but three papillomas arising after long-term TPA promotion (5-7 mo) exhibited c-Ha-ras activation at codon 61 (A-->T and A-->G). Conversely, spontaneous papillomas arising without TPA promotion, including persisting autonomous papillomas, were all negative for activating c-Ha-ras mutations. Both spontaneous and TPA-induced HK1.TGF alpha papillomas expressed c-Ha-ras message levels similar to those in normal, nontransgenic epidermis or HK1.TGF alpha hyperplastic epidermis. These data demonstrate that TGF-alpha overexpression can be an initiating event for TPA promotion, that papillomatogenesis in HK1.TGF alpha mice proceeds frequently via a pathway independent of Ha-ras activation or overexpression, and, thus, that other events are required for autonomous growth and malignant conversion.

Animals↗
PubMed1994

Genetic disorders of keratin: are scarring alopecias a sub-set?

Recent advances have challenged the prevailing view that keratins are merely passive bystanders of keratinocyte biology. With the exciting discovery that three autosomal dominant genetic skin disorders, epidermolysis bullosa simplex (EBS), epidermolytic hyperkeratosis (EHK) and palmoplantar keratoderma (PPK), are in fact disorders of keratins comes the realization that the integrity of the keratin filament network is crucial to the structural integrity of the skin. Since it has been recently established that mutations in keratins K5/K14, K1/K10 and K9 are causative for these keratinocyte disorders, it is very likely that mutations in K6 or in its obligate partner, K16 will result in disease. In order to test this we have produced transgenic mice that express a mutant K6 gene. These mice develop a progressive scarring alopecia at about 6 months of age. Later, the denuded areas developed a keratosis which was prone to infection. Ultrastructural analysis suggests that hair loss is due to the destruction of the outer root sheath. We believe that these mice are models of another keratin disorder.

Alopecia↗
PubMed1994

Mutations in the rod domain of keratin 2e in patients with ichthyosis bullosa of Siemens.

Ichthyosis bullosa of Siemens (IBS) is an autosomal dominant skin disorder that resembles epidermolytic hyperkeratosis (EHK). We have identified mutations in two families originally diagnosed with EHK and in four families diagnosed with IBS at the same codon in the highly conserved carboxy terminal of the rod domain of keratin 2e, thus revealing a mutational hot spot. Our results allow a differential diagnosis to be made between IBS and EHK at the genetic level and we suggest that patients diagnosed with EHK, but lacking keratin K1 or K10 mutations, should be re-examined for mutations in their K2e genes.

Adult↗
PubMed1994

Application of cantharidin or 12-O-tetradecanoylphorbol-13-acetate on mouse epidermis induces a cell population shift that causes altered keratin distribution.

The tumour promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) causes changes in epidermal protein expression, especially in the major differentiation products keratins K1 and K10. These keratins and filaggrin were studied in a pulse-labelled cell cohort in hairless mouse epidermis stimulated to proliferate by TPA or the hyperplasiogen cantharidin. Cells in DNA synthesis were pulse-labelled by 5-bromo-2-deoxyuridine (BrdU) 16 h after topical application of cantharidin or TPA. The BrdU-labelled cell cohort, the two keratins, and filaggrin were spatially mapped by paired immunofluorescence staining. Both cantharidin- and TPA-treated epidermis displayed altered distributions of K1 and K10 with expression only in the outermost cell layers, but the start of their postreplicative expression paralleled that in normal epidermis (18 h for K1 and 24 h for K10 after the last round of DNA synthesis). Cantharidin- and TPA-induced epidermal hyperplasia showed increased basal cell proliferation, accelerated suprabasal migration, and shortened transit time. Thus, the newly formed hyperplastic epidermis was composed of keratinocytes with a lower mean cellular age than that seen in unperturbed epidermis, which caused altered distribution of K1 and K10. Both hyperplastic and normal epidermis showed filaggrin expression in stratum granulosum; this started earlier in treated (30-36 h) than in untreated (96 h) skin. We concluded that the postmitotic onset of K1 and K10 expression was unaltered in regenerative epidermis, whereas filaggrin expression was considerably accelerated and thus influenced by the cell kinetic changes.

Animals↗
PubMed1994

Prenatal diagnosis of epidermolytic hyperkeratosis by direct gene sequencing.

Epidermolytic hyperkeratosis (bullous congenital ichthyosiform erythroderma) is an autosomal dominant skin disorder caused by defects in the suprabasal keratins. Recently, mutations in the keratins 1 and 10 have been identified in patients with this disease. In this study, direct gene sequencing was used to establish the prenatal diagnosis in 15-week gestation twins at risk for epidermolytic hyperkeratosis. Direct sequence analysis of genomic DNA from the affected father and from both chorionic villus samples revealed a tyrosine to asparagine mutation at position 14 within the highly conserved 1A alpha-helical segment of keratin 10. None of the unaffected family members that were analyzed exhibit this mutation nor have polymorphic variations been observed in the normal population at this position. This residue is invariant in all type I keratins sequenced to date and is also conserved in related intermediate filament proteins such as vimentin and lamin. Given this high degree of conservation it is probable that any mutation at this position is deleterious and will result in disease.

Adult↗
PubMed1994

Abnormal keratin 1 and 10 cytoskeleton in cultured keratinocytes from epidermolytic hyperkeratosis caused by keratin 10 mutations.

Epidermolytic hyperkeratosis is caused by mutations of the differentiation-specific keratins K1 and K10. These mutations produce a weakened cytoskeleton that is prone to collapse resulting in cell fragility and lysis. In this study we have analyzed cultured keratinocytes from EHK patients bearing 10R-to-H and 15L-to-S mutations within the 1A segment of the K10 rod domain. Keratinocytes were grown submerged in serum-free medium and induced to differentiate by growing to confluence and increasing the Ca++ concentration in the medium. Cultures were either harvested for mRNA sequence analysis or subjected to immunofluorescence microscopy. Differentiating keratinocytes from these patients were found to express these K10 mutations in their mRNA. Moreover, these cells could be distinguished from normal keratinocytes by their aberrant morphology. EHK keratinocytes frequently exhibited a collapsed perinuclear network of K1/K10 filaments and sometimes peripheral granules of K1 and K10 aggregates, reminiscent of the cells of the suprabasal layers in these patients. This report documents the expression of mutant keratin 10 in cultured EHK keratinocytes.

Base Sequence↗
PubMed1994

Full-thickness skin grafts from flaky skin mice to nude mice: maintenance of the psoriasiform phenotype.

Flaky skin (fsn) is an autosomal recessive mouse mutation with papulosquamous disease features similar to human psoriasis. In fsn/fsn skin, one sees marked acanthosis and hyperkeratosis with focal parakeratosis, subcorneal pustules, dermal capillary dilation, and a marked diffuse dermal infiltration of mixed inflammatory cells, predominantly lymphocytes. To determine if these pathologic features are a characteristic of the skin or a chronic autoimmune attack, we placed full-thickness skin grafts from affected homozygous (fsn/fsn) and normal littermate control (+/?) mice on the dorsal skin of genetically athymic nude (nu/nu) mice. After 10 weeks of observation, the grafts maintained the histologic phenotype of the donor animal. In the fsn/fsn grafts, there was persistence of both epidermal proliferation and dermal inflammation, characteristics of the mutation. The fsn/fsn phenotype was also confirmed by immunohistochemical evaluation for specific mouse keratinocyte marker expression. Based on tritiated thymidine uptake, we found DNA synthesis rates elevated threefold or more in fsn/fsn epidermis compared to littermate control mouse skin. Elevated rates of DNA synthesis remained a feature of the fsn/fsn grafts but not that of littermate control skin grafts. This study demonstrates that the psoriasiform phenotype of this mouse mutation can persist independent of the host thymic-derived immune system.

Animals↗
PubMed1994

Retinoic acid suppression of loricrin expression in reconstituted human skin cultured at the liquid-air interface.

Retinoids have been shown to modulate the expression of proteins involved in epidermal differentiation. To examine this effect in an in vitro skin model, we evaluated the effect of retinoic acid on the expression of two cell envelope proteins, loricrin and involucrin, and an early marker of epidermal differentiation, keratin 1, in a reconstituted human skin equivalent cultured at the liquid-air interface. Retinoic acid, a known inhibitor of keratinization in monolayer and raft cultures, was evaluated for its ability to alter the expression and distribution of these markers of epidermal differentiation. Retinoic acid (10(-6) M) suppressed loricrin expression in skin cultures as determined by both protein and mRNA analysis. In contrast, no inhibition of involucrin or K1 expression was observed at the protein level at the same retinoic acid concentration. However, some suppression of K1 mRNA transcription was observed in retinoic acid-treated cultures. These results demonstrate that in differentiating cultures of reconstituted human skin, loricrin expression is markedly inhibited by retinoids, K1 less so, and involucrin not at all.

Base Sequence↗
PubMed1994

Epidermis: an attractive target tissue for gene therapy.

Important advances have been made within the past several years in understanding diseases at the molecular and cellular levels, which may enable the application of somatic gene therapy to a wide variety of genetic and acquired diseases. The initial clinical trials involving somatic gene therapy have demonstrated that gene transfer into human subjects can be performed safely and with public acceptance. This review focuses on use of the epidermis as a target tissue for gene therapy and assesses various delivery systems for both ex vivo and in vivo approaches. In addition, we discuss candidate diseases that may be amenable to epidermal gene therapy and the advantages of employing transgenic mouse model systems to test the efficacy of a given gene therapy prior to clinical trials.

Animals↗
PubMed1994

Transgenic mice expressing targeted HPV-18 E6 and E7 oncogenes in the epidermis develop verrucous lesions and spontaneous, rasHa-activated papillomas.

In order to create a transgenic model for human papilloma virus (HPV)-associated carcinogenesis, we have used the regulatory elements of a human keratin K1 (HK1) gene to target the expression of the E6 and E7 oncogenes of HPV-18 exclusively to the epidermis. All murine expressors were viable and lived normal lifetimes; older mice (> 1 year) possessed numerous small lesions with a verrucous (wart-like) histotype. Analysis of newborn epidermis and lesions revealed that the HPV-18 E6/E7 genes were being expressed with a predominance of the E6*/E7 transcript over the full length E6/E7 message. The long latency in lesion appearance may reflect the low level of intact E6 transcripts and the requirement for additional genetic or epigenetic events before production of an overt lesion. In agreement with this proposal, spontaneous papillomas developed that expressed an activated rasHa oncogene (codon 61, A-->T; codon 13, G-->T). All lesions expressed keratin genes K1, K6, and K13 in a fashion characteristic of hyperproliferative or benign tumors with no evidence of malignant conversion. Our results demonstrate that the mouse epidermis represents a relevant in vivo model system to analyze the interaction between HPV and cellular genes in neoplasia.

Animals↗