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D R Roop

Publications and source records attributed to D R Roop.

At least 109 records · Page 6Linked to original sources
PubMed1991

Transcription of the human loricrin gene in vitro is induced by calcium and cell density and suppressed by retinoic acid.

We have previously shown that loricrin is a major component of the cornified cell envelope (CE) expressed late in epidermal differentiation in the granular layers of normal skin. Normal human keratinocytes were cultured under various conditions and loricrin mRNA levels were assessed at various time points. Only Ca++ concentrations above 0.1 mM Ca++ were permissive for the expression of lori-crin mRNA. Maximal mRNA levels were found at 0.35 mM Ca++ and a critical cell density appeared to be required for optimal accumulation of loricrin transcripts. Retinoic acid (RA) at 10(-7) to 10(-9) M completely blocked Ca+(+)-induced loricrin mRNA synthesis when applied simultaneously. Furthermore, addition of RA to cultures already exposed to higher Ca++ levels resulted in the complete loss of loricrin mRNA within 48-72 h. So far, no other components of the CE have been shown to be suppressed by RA. However, similar patterns of expression were reported for filaggrin, a matrix protein also expressed late in epidermal differentiation. Therefore, we compared the mRNA levels of loricrin and filaggrin and found them to change in parallel in response to the various culture conditions. These results suggest that Ca++, cell density, and RA are crucial regulators of loricrin expression in vitro and that the transcriptional control of loricrin and filaggrin expression in the epidermis are closely coordinated.

Blotting, Northern↗
PubMed1991

Myoepitheliomas in inbred laboratory mice.

Myoepitheliomas are subcutaneous tumors that arise from myoepithelial cells of various exocrine glands. In a retrospective study of 142 tumors observed over a period of 3 years, myoepitheliomas occurred spontaneously in A/HeJ, A/J, BALB/cJ, BALB/cByJ, LLC.A/Ckc, and NOD/Lt inbred strains of mice. Tumors presented primarily in the subcutaneous tissues of the ventral neck (74% of the myoepitheliomas evaluated) but were observed in several other subcutaneous locations, including the head, perineum, and ventral abdomen. These areas were adjacent to salivary, mammary, clitoral, preputial, and Harderian glands. Forty myoepitheliomas were tested by the avidin-biotin complex technique with a panel of antisera specific for mouse keratins, intermediate filaments, and other cytoskeletal proteins to determine the cell type from which this neoplasm originated. Antibodies directed against the specific mouse keratins K5, K6, and K14, and a broadly cross-reactive cytokeratin antibody stained acinar and ductal myoepithelial cells in normal mammary, salivary, and Harderian glands, and neoplastic cells in all cases. Antisera directed against a smooth muscle actin (anti-alpha-sm-1) stained acinar myoepithelial cells of the glands and vascular smooth muscle but neither ductular myoepithelial cells nor tumor cells. This supports the notion that these tumors originate from extraglandular ductular myoepithelial cells. Southern blots, prepared from DNA extracted from nine myoepitheliomas, did not show restriction fragment length polymorphisms when mouse mammary tumor virus (MMTV) cDNA or Int-1 genomic DNA probes were used; this implies that a retrovirus is not the etiologic agent.

Age Factors↗
PubMed1991

A human epidermal differentiation-specific keratin gene is regulated by calcium but not negative modulators of differentiation in transgenic mouse keratinocytes.

Keratins K1 and K10 represent the major differentiation products of the maturing epidermal keratinocytes. Primary epidermal cell cultures from newborn K1 transgenic mice containing a 12-kilobase human K1 genomic fragment were established in order to examine the expression of both human and mouse K1 in the presence of known modulators of epidermal differentiation. Elevated levels of Ca2+ in the culture medium induced both mouse K1 and human K1. Supplementing the medium with retinoic acid or 12-O-tetradecanoylphorbol-13-acetate or introducing a Harvey viral ras oncogene (v-rasHa) into the cells completely suppressed mouse K1 but not human K1. Our results suggest that: (a) the human 12-kilobase insert contains all the necessary cis-acting elements to respond to the Ca2+ signal, and (b) other cis-acting elements, not present within this insert, may function independently to regulate the response of K1 to retinoids, 12-O-tetradecanoylphorbol-13-acetate, and v-rasHa transformation. This transgenic model provides an approach to identify elements required for the regulation of an epidermal differentiation-specific gene.

Animals↗
PubMed1990

Changes in keratin expression during 7,12-dimethylbenz[a]anthracene-induced hamster cheek pouch carcinogenesis.

This study was undertaken to explore the expression of keratins in the hamster cheek pouch carcinogenesis model, using monospecific keratin antibodies and a technique that allows immunoblotting analysis of tissues embedded in paraffin. Changes in keratin expression were correlated with histopathological changes and with the expression of the enzyme gamma-glutamyl transpeptidase. The right cheek pouch of 20 male golden Syrian hamsters was treated with 0.5% 7,12-dimethylbenz[a]anthracene for 16 weeks. As previously described by other laboratories, this treatment resulted in hyperplastic and dysplastic lesions and benign and malignant tumors. The keratins assayed in this study were K14 (Mr 55,000), K1 (Mr 67,000), and K13 (Mr 47,000). The normal hamster cheek pouch epithelium expressed K14 in the basal layer and K13 in the suprabasal and differentiated layers, whereas K1 was not detected by either immunohistochemistry or immunoblotting. Concomitant with 7,12-dimethylbenz[a]anthracene-induced hyperplasia, there were some topographical alterations in the distribution of K14. In this case, K14 was no longer restricted to the basal layer but was also expressed in differentiated cells. The same pattern was also observed in dysplastic lesions and in squamous cell carcinoma. Furthermore, expression of the K13 differentiation-associated keratin was preserved in this hyperplastic epithelium during all the stages of carcinogenesis, including either anaplastic or differentiated areas. In contrast, after 2 weeks of 7,12-dimethylbenz[a]anthracene treatment, K1 expression started as a weak and patchy pattern in suprabasal cells, becoming stronger and more homogeneous at 8 and 16 weeks of treatment. However, K1 was almost absent in squamous cell carcinoma, where only small very well differentiated areas were stained. We also observed gamma-glutamyl transpeptidase-positive foci in earlier stages of carcinogenesis, concomitant with the expression of the K1 keratin. However, it was not possible to find a perfect topographical correspondence between the two events. Alterations in the pattern of keratin expression appear to be a common feature during the development of squamous cell carcinoma in different systems and could be an excellent tool to study carcinogenesis and chemoprevention.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1990

Two-colour immunofluorescence marker study of pleomorphic adenomas.

Extensive use of two-colour immunofluorescence staining for various cell markers in pleomorphic adenoma, revealed three consistent phenotypic features: (1) keratin polypeptide No. 14, which was virtually restricted to myoepithelial cells (MEC) in normal salivary glands, appeared in a large fraction of the tumour cells, suggesting that the principal neoplastic element is derived from MEC or their immediate precursors; (2) a complex co-expression pattern of various cell markers was found, with extensive concurrence of keratin and vimentin in strands of MEC-like and myxoid tumour cells, probably reflecting different degrees of tumour cell differentiation; and (3) two phenotypically distinctive dendritic cell populations were identified, one consisting of keratin positive tumour cells and the other of HLA-DR positive but keratin negative stromal cells. The significance of these findings with regard to the histogenesis and complex morphology of pleomorphic adenoma is discussed.

Adenoma, Pleomorphic↗
PubMed1990

Early expression of type I K13 keratin in the progression of mouse skin papillomas.

The premalignant evolution of chemically induced mouse skin papillomas is characterized by dysplastic changes, aneuploidy, induction of gamma-glutamyl transpeptidase (GGT), and changes in the expression of keratins, especially differentiation-associated K1. This keratin, which is expressed in normal epidermis and early papillomas, is no longer present in more advanced dysplastic and aneuploid papillomas and in fully invasive carcinomas. More recently, it has been shown that K13, a keratin normally present in internal epithelia but not in epidermis, is aberrantly expressed in epidermal tumors. In the present study, the timing of expression of K13 and its correlation with other markers of premalignant evolution were investigated. Papillomas were induced by SENCAR mice by a single initiating dose of 20 nmol of 7,12-dimethylbenz[a]-anthracene (DMBA) and promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA) (2 micrograms twice a week). Tumors were randomly harvested at 10, 20 and 35 weeks of promotion. K13 and K1 expression in papillomas was studied using immunoblotting and immunostaining of consecutive sections, as previously described. As expected from previous studies, the distribution of K1 in papillomas collected at 10 weeks of promotion was restricted to differentiated cells and was uniform throughout the section of the papilloma. Conversely, K13 was expressed only as small foci in 10 out of 21 papillomas (48%). Papillomas of 20 weeks were also positive for K1. Staining for K13 was positive in these papillomas with the exception of only one that was essentially negative, presenting only one small positive focus. Some of the papillomas collected at week 35 were negative for K1, but immunostaining with K13 showed uniform staining of suprabasal cells in all the papillomas studied. In all cases, immunohistochemical results were confirmed by immunoblotting with proteins extracted from 7 microns sections from each paraffin block. These results indicate that keratins K1 and K13 are coexpressed in most papillomas from 10 to 35 weeks of promotion. However, analysis of adjacent sections showed that K13 positive areas are topographically located in the K1 negative areas of the papillomas, suggesting a shift in the differentiation program from epidermal to mucosal types of keratinization. Based on these and previous studies from our laboratory, we conclude that K13 is an early marker of papillomas progression, which occurs before gross chromosomal abnormalities are present in the stem line of the tumors, and precedes dysplastic changes and the onset of GGT expression, and is probably concomitant at the individual cell level with loss of K1.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1990

Changes in photo-aged human skin following topical application of all-trans retinoic acid.

Although topical applications of retinoids on rodents and humans have been shown to cause epidermal hyperplasia, a detailed study of the influence of retinoids on epidermal differentiation in vivo has not been performed. In order to assess the pharmacologic effects of chronic topical tretinoin application used to improve the appearance of patients with photoaged skin, cutaneous biopsies from 25 patients in a controlled clinical study were examined histologically and immunocytochemically. Chronic application of tretinoin causes epidermal thickening (25 of 25 samples), stratum granulosum thickening (15 of 25), parakeratosis (13 of 25), a marked increase in the number of cell layers expressing epidermal transglutaminase (13 of 25), and focal expression of two keratins, K6 (12 of 25) and K13 (8 of 25), not normally expressed in the epidermis. The morphologic changes correlated with immunohistochemical abnormalities; neither of these correlated with the subjective cosmetic response. Three major epidermal differentiation products, keratins K1, K10, and K14 were not altered, within the limits of the methods used. Thus, chronic topical tretinoin reprograms some, but not all, aspects of human epidermal differentiation in vivo.

Administration, Topical↗
PubMed1990

Targeting gene expression to the epidermis of transgenic mice: potential applications to genetic skin disorders.

The ability to specifically target gene expression to the epidermis of transgenic mice offers the exciting possibility of creating animal models of certain skin disorders that are inherited in man. It may be possible to produce mouse models of dominantly inherited keratinization disorders by targeting the expression of mutant genes encoding the major differentiation products of the epidermis, such as the differentiation specific keratins, filaggrin and cell envelope proteins. Mouse models for other skin disorders associated with abnormal regulation of growth, such as psoriasis, may be generated by targeting the overexpression of cytokines and growth factors, which are thought to play important roles in the pathogenesis of this disease. The development of currently unavailable animal models for certain inherited human skin diseases would not only contribute to our understanding of the pathogenesis of these diseases at the molecular level, but also provide interesting models for therapeutic intervention.

Animals↗
PubMed1990

Differential keratin gene expression in developing, differentiating, preneoplastic, and neoplastic mouse mammary epithelium.

Two keratins whose expression has been associated with proliferation (K14) and hyperproliferation (K6) in mouse epithelia were detected in normal, preneoplastic, and neoplastic mouse mammary tissues. K6 and K14 keratins were independently expressed in distinct epithelial cell populations in developing mammary anlage. K6 was confined to a small number of mammary epithelial cells associated with the growing end buds and among the proximal luminal epithelium, whereas K14 expression appeared in basally located fusiform cells that correspond to the location of mammary myoepithelial cells. This pattern was maintained in mature glands and through full functional differentiation with the exception that K6-positive cells were only rarely detectable. During lobuloalveolar growth in early pregnancy, K6 and K6/K14 coexpressing cells were observed among the luminal and suprabasal cells in the expanding lobular epithelium. This K6/K14 coexpressing epithelial subset persisted throughout pregnancy, lactation, and involution, albeit in much smaller numbers than observed in early pregnancy. Two patterns of K6 and K14 expression in preneoplastic and neoplastic lesions of mouse mammary glands were induced by various carcinogenic stimuli. In one, increased numbers of K6- or K14-positive cells were present in distinct cellular populations; in the other, coexpression of K6/K14 was found in a large subpopulation of both preneoplastic and neoplastic mammary epithelium. These observations suggest that expression of K6 and K14 keratins in the mouse mammary gland is associated with growth and expansion of specific mammary epithelial cell populations, and as such these keratins may be useful probes with which to identify mammary epithelium-specific primordial cells. In agreement with this possibility, K6/K14 expression was demonstrated within a distinct subset of morphologically distinct luminal mammary epithelial cells that have been reported to possess kinetic properties in vitro consistent with those expected of latent mammogenic stem cells.

Animals↗
PubMed1989

Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentrations in vitro.

Epidermal differentiation is characterized by a series of coordinated morphological and biochemical changes which result in a highly specialized, highly organized, stratified squamous epithelium. Among the specific markers expressed in differentiating epidermis are (a) two early spinous cell proteins, keratins 1 and 10 (K1 and K10); and (b) two later granular cell proteins, filaggrin and a cornified envelope precursor (CE). In vitro, epidermal basal cells are selectively cultured in 0.05 mM Ca2+ medium, and terminal differentiation is induced when the Ca2+ concentration is increased to 1 mM. However, only a small fraction of the cells express the markers K1, K10, CE, or filaggrin in the higher Ca2+ medium. To explore the factors required for marker expression, cultured epidermal cells were exposed to intermediate Ca2+ concentrations and extracts were analyzed using specific antibody and nucleic acid probes for the four markers of interest. These studies revealed that marker expression was enhanced at a restricted concentration of Ca2+ in the medium of 0.10-0.16 mM. At this Ca2+ concentration, both protein and mRNA levels for each marker were substantially increased, whereas at higher or lower Ca2+ concentrations they were diminished or undetected. The percentage of cells expressing each marker was increased two- to threefold in the permissive Ca2+ medium as determined by immunofluorescence analysis. This optimal level of Ca2+ was required both to initiate and sustain marker expression. At the permissive Ca2+ concentration, expression of the markers was sequential and similar to the order of appearance in vivo. K1 was expressed within 8-12 h and K10 was expressed in the ensuing 12-24-h period. CE and filaggrin were expressed in the subsequent 24 h. Inhibition of K1 expression by cycloheximide suggested that an inducible protein was involved. Other investigators have determined that a shallow Ca2+ gradient exists in epidermis, where the basal cells and spinous cells are in a Ca2+ environment substantially below serum Ca2+ levels. These in vitro results suggest that the Ca2+ environment is a fundamental regulator of expression of epidermal differentiation markers and provide an explanation for the existence of the Ca2+ gradient in vivo.

Animals↗
PubMed1988

The complete sequence of the human intermediate filament chain keratin 10. Subdomainal divisions and model for folding of end domain sequences.

We present the complete amino acid sequence of the human keratin 10 (type I) intermediate filament chain expressed in terminally differentiated epidermal cells. Comparisons of this sequence with its mouse and bovine counterparts allow us to describe structural features of the functional end domains. First, sections of their respective end domains are highly conserved and permit a redefinition of earlier models for their subdomainal organization. The amino-terminal end domain consists of El, the first 57-58 residues that are basic, glycine-rich, and have been highly conserved among the three species; V1, a region of well-defined quasi repeats of the motif aliphatic-serine/glycinen; and H1, a newly recognized short acidic sequence that has been conserved among the type I keratin family. The carboxyl-terminal end consists of V2 and E2 whose properties but not sequence resemble V1 and E1, respectively. Second, since the E1, H1, and E2 sequences have been highly conserved between the three species, we suggest they are critical elements in defining intermediate filament function. Third, we note that the E and V sequences of the keratin 10 (and other keratin) chains share many properties in common with protein chain turns found in globular proteins. We therefore propose a model in which these sequences form omega loop-like structures (Leszczynski, J. N. & Rose, G. D. (1986) Science 234, 849-855) on the surface of keratin intermediate filaments. This represents the first specific proposal for the end domain structure of any intermediate filament chain.

Amino Acid Sequence↗
PubMed1988

Transcriptional control of high molecular weight keratin gene expression in multistage mouse skin carcinogenesis.

Monospecific antikeratin antisera and specific complementary DNA probes were used to analyze expression of keratin genes in newborn mouse skin and skin papillomas and carcinomas by indirect immunofluorescence, immunoblotting, and in situ hybridization. Tumors were induced by initiation with 7,12-dimethylbenz[a]anthracene and promotion with 12-O-tetradecanoylphorbol-13-acetate. Type I epidermal keratin K14 protein (Mr 55,000) is found in all living layers of the newborn skin but is most abundant in the lower strata. K1 (Mr 67,000) and K10 (Mr 59,000) proteins are predominantly suprabasal and K1 is processed in the stratum corneum. Transcripts for K14 were confined largely to the basal cell layer by in situ hybridization. Transcripts for K1 and K10 were highly expressed in suprabasal cells including the granular cell layer. In benign tumors, distribution of K14 protein is similar to that in newborn skin, while the abundance of K1 and K10 appears to be somewhat reduced although the tissue distribution remains suprabasal. Transcription of K14 is aberrant in benign tumors and transcripts persist throughout much of the suprabasal cell layers. Transcripts of K1 and K10 are normally distributed in papillomas but grain density is less intense than in newborn epidermis. Keratin expression in carcinomas is highly disturbed. K14 protein and transcripts are highly expressed in all strata in carcinomas while protein and transcripts for K1 and K10 are essentially absent. These results suggest that papilloma cells fail to respond to or generate signals to regulate K14 expression in the differentiating suprabasal cell layers and may not fully express their suprabasal cell keratins. Carcinomas fail to express suprabasal cell keratins and this is regulated at the transcriptional level. The loss of suprabasal keratin expression may provide a marker for malignant conversion in the mouse skin carcinogenesis model.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1988

Sequential development of aneuploidy, keratin modifications, and gamma-glutamyltransferase expression in mouse skin papillomas.

To elucidate the role and timing of expression of different premalignant and malignant markers in tumor promotion, we correlated alterations in keratin patterns and gamma-glutamyltransferase (GGT) expression with the chromosomal status of individual mouse skin papillomas. Papillomas were induced by 7,12-dimethylbenz[a]anthracene initiation and 12-O-tetradecanoylphorbol-13-acetate promotion. Individual tumors were randomly sampled at 20 and 35 weeks of promotion. Each tumor was cytogenetically analyzed and serial paraffin sections were used for GGT detection, immunoblotting, and immunohistochemistry studies. Monospecific antibodies elicited against keratins K1 (Mr 67,000) and K14 (Mr 55,000) were used to analyze keratin modifications. Most tumors at 20 weeks of promotion, although exhibiting aneuploidy, still presented high levels of the K1 differentiation-associated keratin. Later during promotion those tumors bearing the highest aneuploidy indexes were those that showed a marked decrease in or absence of the K1 protein. Furthermore, those same tumors with the highest levels of genomic alterations also exhibited foci of GGT activity. These results support the idea that the majority of papillomas under continuous promotion are progressing toward malignancy. Aneuploidy seems to precede detectable keratin modifications, and GGT activity appears to be the latest marker to be expressed.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1988

The expression and modulation of IL-1 alpha in murine keratinocytes.

Murine and human keratinocytes produce an IL-1-like factor that appears to be similar if not identical to monocyte-derived IL-1. IL-1 may be an important mediator in cutaneous inflammatory responses, however, little is currently known concerning factors that may modulate IL-1 expression in keratinocytes. To address this issue we examined the effect of LPS, UV, and the cell differentiation state on murine keratinocyte IL-1 mRNA expression. Our results indicated that as with the murine P388D1 monocyte cell line, PAM 212 keratinocytes constitutively express abundant amounts of IL-1 alpha mRNA. On exposure to LPS (100 micrograms/ml) for 8 h there was more than 10 times the increase in PAM 212 IL-1 alpha mRNA which was accompanied by a sixfold increase in supernatant IL-1 activity. Similarly UV irradiation had a significant effect on keratinocyte IL-1 alpha expression. High dose UV (300 mJ/cm2) inhibited PAM 212 IL-1 alpha expression at 4, 8, 24, 48 h post-UV whereas a lower dose of UV (100 mJ/cm2) inhibited UV at 4 and 8 h post-UV, but induced IL-1 expression at 24 and 48 h post-UV. The expression of IL-1 alpha varied with the differentiation state of the keratinocytes. Freshly removed newborn murine keratinocytes were found to constitutively express IL-1 alpha mRNA. Keratinocytes grown in low [Ca2+] tissue culture media (0.05 mM) for 6 days, functionally and phenotypically become undifferentiated and express increased quantities of IL-1 alpha mRNA, whereas cells grown in high [Ca2+] media (1.2 mM) for 6 days become terminally differentiated and IL-1 expression ceased. Keratinocytes cultured for 3 days in low [Ca2+] conditions expressed an intermediate level of IL-1 alpha. In contrast, little or no IL-1 beta mRNA was detected in either the PAM 212 cells or newborn murine keratinocytes. Thus LPS, UV, and cell differentiation state have a significant effect on expression of IL-1 alpha in murine keratinocytes.

Animals↗
PubMed1988

Aberrant expression during two-stage mouse skin carcinogenesis of a type I 47-kDa keratin, K13, normally associated with terminal differentiation of internal stratified epithelia.

Specific keratin cDNA probes and monospecific antikeratin antisera were used to analyze mouse epidermis and epidermal tumors for the expression of a type I 47-kDa keratin, K13, normally associated with terminal differentiation of internal stratified epithelia. We demonstrated that this keratin was virtually absent from the entire body epidermis at various stages of development. Also, it was not detected in various forms of acute and chronic epidermal hyperproliferation or in epidermal cells cultured under conditions that favored either cell proliferation or in vitro differentiation. In contrast, K13 was consistently expressed in squamous cell carcinomas of the skin induced by 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate (TPA), whereas papillomas obtained by the same two-stage protocol were distinctly heterogeneous with regard to the expression of this keratin. These findings were true for two different strains of mice (NMRI and Sencar). Papillomas collected from Sencar mice after 12 wk or from NMRI mice after 15 wk of promotion with TPA were either negative for K13 or elicited variable amounts of this keratin. In all cases of positive expression of K13 in tumors, as in normal stratified internal epithelia, both the keratin protein and its mRNA invariably occurred in the differentiating cell compartments. In contrast to what we found in internal stratified epithelia, however, K13 was expressed without its commonly encountered type II 57-kDa partner, K4. Papillomas negative for the K13 protein were also devoid of K13 transcripts. This indicates that the aberrant K13 expression in tumors is regulated at the level of transcription. Our results suggest that K13 may provide a marker for malignant conversion in the mouse two-stage skin carcinogenesis model and may be especially suited for studies of gene expression regulation.

Animals↗