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D Roop

Publications and source records attributed to D Roop.

15 recordsLinked to original sources
PubMed1998

Interdependence of cortical thymic epithelial cell differentiation and T-lineage commitment.

Thymocyte and thymic epithelial cell (TEC) development are interdependent processes. Although lineage relationships among progressively maturing thymocyte subsets have been characterized, the developmental relationships among TEC subsets are obscure. Because epithelial cells express distinct keratin (K) species as a function of differentiation stage and proliferative status, we used K expression patterns to identify mouse TEC subsets and determine their lineage relationships. As expected, cortical and medullary TEC subsets express distinct K expression patterns in the normal thymus. However, we detected two distinct cortical TEC subsets, a major K8(+)K5(-) subset and a minor K8(+)K5(+) subset, which is highly represented at the cortico-medullary junction. Both cortical TEC subsets are also present in recombination activating gene 1 (RAG-1(-/-)) and TCRbetaxdelta-/- thymi in which T-cell development is blocked at the CD4(-)CD8(-)CD25(+)CD44(-) pre-T cell stage. In contrast, K8(+)K5(+) TECs predominate in the thymi of human CD3epsilon transgenic mice in which thymocyte development is blocked at an earlier CD4(-)CD8(-)CD25(-)CD44(+) stage. Transplantation of newborn human CD3epsilon transgenic thymi under the kidney capsule of RAG-1(-/-) mice results in the emergence of K8(+)K5(-) TECs concomitant with the appearance of CD25(+) thymocytes. Together, the data suggest that cortical TEC development proceeds from a K8(+)K5(+) precursor subset to a K8(+)K5(-) stage in a differentiation process concomitant with T-cell lineage commitment.

Animals↗
PubMed1998

Human keratin-1.bcl-2 transgenic mice aberrantly express keratin 6, exhibit reduced sensitivity to keratinocyte cell death induction, and are susceptible to skin tumor formation.

Nonmelanoma skin cancers (NMSC) are among the most common malignancies in the world. Typically, these neoplasms grow slowly and are comparatively indolent in their clinical behavior. The most frequent molecular alterations implicated in the pathogenesis of these neoplasms involve genes known to be regulators of cell death including p53, Ha-ras and bcl-2. In order to evaluate the significance cell death deregulation during skin carcinogenesis, we generated a transgenic mouse model (HK1.bcl-2) using the human keratin 1 promoter to target the expression of a human bcl-2 minigene to the epidermis. Transgenic HK1.bcl-2 protein was expressed at high levels specifically in the epidermis extending from the stratum basale through the stratum granulosum. The epidermis of HK1.bcl-2 mice exhibited multifocal hyperplasia without associated hyperkeratosis and aberrant expression of keratin 6. The rate of proliferation was similar in HK1.bcl-2 and control epidermis although suprabasal BrdUrd incorporating cells were present only in HK1.bcl-2 skin. Keratinocytes from the HK1.bcl-2 mice were significantly more resistant to cell death induction by U.V.-B, DMBA, and TPA, compared to control keratinocytes. Furthermore, papillomas developed at a significantly greater frequency and shorter latency in the HK1.bcl-2 mice compared to control littermates following initiation with DMBA and promotion with TPA. Together these results support a role for bcl-2 in the pathogenesis of NMSC.

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

Expression of keratins in mouse vaginal epithelium.

In the epithelium of the rodent vagina proliferation and differentiation are tightly regulated by ovarian hormones. Estrogens stimulate proliferation and squamous differentiation, whereas progesterone redirects differentiation to a mucus-secreting epithelium formed by goblet-like cells. In the present study, we used monospecific keratin antibodies to show the expression and distribution of keratins in SENCAR mouse vaginal epithelium in different stages of the estral cycle and in ovariectomized animals. In ovariectomized animals, the vaginal epithelium expressed K6, K8, K13 and K14, but not K1. After estrogen treatment, K1 was expressed. During proestrus and estrus, the keratin pattern was essentially identical to that observed in 17 beta-estradiol-stimulated animals. In contrast, during the progestational stages (metaestrus and diestrus) or after progesterone treatment of ovariectomized mice, the most relevant change was the loss of K1. Together, these results show that K1 expression is induced by estrogens in the vaginal epithelium. In contrast, K6, K8, K13 and K14 are constitutively expressed even when squamous differentiation is not observed.

Animals↗
PubMed1993

Low frequency of codon 61 Ha-ras mutations and lack of keratin 13 expression in 7,12-dimethylbenz[a]-anthracene-induced hamster skin tumors.

Alterations in the pattern of keratin expression are a common feature of skin-tumor development. In this study, we investigated whether the loss of epidermal keratin 1 (K1) and its replacement by mucosal keratin 13 (K13) is unique to mouse skin tumors induced by 7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA), since it has been reported that human epidermal tumors do not exhibit aberrant expression of K13. With that purpose, we analyzed the keratin profiles of 16 DMBA-induced hamster skin tumors using monospecific antibodies against K1 and K13. Although all the tumors expressed K1, they also showed an overall tendency towards loss of this keratin; furthermore, none of the tumors expressed K13. Previous studies have suggested that the induction of K13 in mouse skin is related to the mutation of the Ha-ras gene by the initiating agent DMBA, a mutation consistently found in murine DMBA/TPA-induced tumors and rarely found in human skin tumors. Therefore, we also evaluated the tumors for the presence of codon-61 mutations by direct sequencing of DNA extracted from paraffin-embedded tissue sections. Only three tumors showed an A-->T transversion in the second nucleotide of Ha-ras codon 61. However, presence of the mutation did not correlate with K1 staining. Although hamster skin tumors were induced by the same initiator as were mouse skin tumors, hamster skin tumors did not show the same keratin profile. Moreover, their immunohistochemical expression of K1 and K13 and their codon 61 sequences resembled that of their human counterparts. These results suggest that the aberrant expression of K13 may be unique to murine skin. Furthermore, although codon 61 Ha-ras mutation appears to be related to keratin alterations in the mouse model, this mutation is not sufficient to produce the same biochemical changes in other species.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1991

Altered regulation of growth and expression of differentiation-associated keratins in benign mouse skin tumors.

Alterations in the pattern of epidermal cell differentiation and proliferation in mouse skin and benign skin tumors were studied by two-color immunofluorescence using monospecific antibodies. Replicating cells were identified by 5-bromo-deoxyuridine (BrdU) pulse-labeling and differentiating cells by keratins K1 and K10. In normal mouse skin, pulse-chase experiments for 120 h revealed that replication was restricted to a single layer of basal cells. Replicating cells did not express K1 or K10, but these keratins were sequentially expressed in post-mitotic basal cells 18 and 24 h following DNA synthesis respectively, and cells expressing these keratins migrated into the suprabasal layers. In phorbol-ester- or cantharidin-stimulated hyperplastic skin, replicating cells were also confined to the basal cell compartment and suprabasal cells expressed keratins 1 and 10. In papillomas induced by initiation with 7,12-dimethylbenz[a]anthracene and promotion with 12-O-tetradecanoylphorbol-13-acetate, replication occurred predominantly in cells in an expanded basal cell compartment (two to four layers above the basement membrane). Cells in these basal layers did not express K1 or K10, but more superficial cells did. After a 1 h pulse of BrdU, replication was also identified in suprabasal cells expressing the differentiation-associated keratins. These and other results suggest that benign tumor cells escape the obligatory growth arrest associated with differentiation. Replication of K1- and K10-expressing suprabasal cells may represent an early alteration during mouse skin carcinogenesis.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1991

Alterations in epidermal biochemistry as a consequence of stage-specific genetic changes in skin carcinogenesis.

The induction of cancer on mouse skin by initiation-promotion protocols occurs through stages in which a benign squamous papilloma is an obligate precursor of squamous cell carcinoma. Activation of the Ha-ras gene is sufficient to produce the papilloma phenotype, while additional genetic changes are required for malignant conversion. The introduction of Ha-ras into normal keratinocytes suppresses the expression of differentiation markers, keratin K1 and K10, and loricrin (a cornified envelope precursor) and, to a lesser extent, filaggrin, at the level of transcription. However, cells initiated by Ha-ras express a nonepidermal keratin, K8. The transcription of K8 in these cells is sensitive to the level of medium Ca2+, being abundant in 0.5 mM Ca2+ and not detected in 0.05 mM Ca2+. Epidermal differentiation is regulated by signalling, which involves changes in phosphatidylinositol turnover and intracellular Ca2+. Cells initiated by Ha-ras do not differ from normal keratinocytes in their intracellular Ca2+ response patterns, at least in response to changes in extracellular Ca2+ and serum factors. However, c-Ha-ra keratinocytes have a high basal level of phosphatidylinositol (PI) turnover, which is additive with several other inducers of this pathway, including Ca2+ and aluminum fluoride. Additional studies suggest that high turnover of the PI pathway is incompatible with differentiation-specific gene expression in keratinocytes. We suggest this negative relationship is mediated through elevated diacylglycerol production and chronic down-modulation of protein kinase C. Protein kinase C is known to be essential for expression of differentiation-related genes in keratinocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

The v-ras oncogene inhibits the expression of differentiation markers and facilitates expression of cytokeratins 8 and 18 in mouse keratinocytes.

Cultured mouse keratinocytes can be initiated in vitro by the introduction of a v-rasHa gene by viral transduction. Previous studies indicated that v-rasHa-transduced keratinocytes have a high proliferation rate in medium with 0.05 mM Ca2+ and resist terminal differentiation in medium with greater than 0.1 mM Ca2+, a culture condition in which normal cells mature into squames. The current studies demonstrate that v-rasHa keratinocytes do not express transcripts or protein for epidermal early differentiation markers keratins 1 and 10 when cells are challenged with 0.12 mM Ca2+, which is a signal for expression of these genes in normal cells. Both transcript and protein for the late differentiation marker loricrin are also diminished in v-ras keratinocytes, but filaggrin, also a late differentiation-related gene product, is expressed in nearly normal amounts but at a different Ca2+ optimum. Modification of intracellular Ca2+ with ionomycin failed to restore the expression of any suprabasal keratinocyte markers. In contrast to the effects on normal products of keratinocyte differentiation, the introduction of the v-rasHa gene facilitated the expression of keratins 8 (K8) and 18 (K18). These keratins are characteristic of embryonic cells and cells of simple adult epithelia but not stratified squamous epithelia such as skin. Like normal differentiation markers, the expression of K8 and K18 was dependent both on the v-ras oncogene and the Ca2+ concentration of the culture medium, with greater than 0.1 mM Ca2+ being optimal. At the optimal Ca2+ level, the majority of v-ras keratinocytes expressed K8 and K18 after 96 h, and many cells had reduced amounts of the normal keratinocyte cytokeratin K14. These studies indicate that the v-ras gene causes substantial reprogramming of epidermal physiology, producing an unusual phenotype devoid of early suprabasal markers but at least partially permissive for late marker expression. Furthermore, the Ca2(+)-dependent expression of K8 and K18 suggests that a normal signalling pathway used in keratinocyte differentiation is diverted to an abnormal endpoint.

Animals↗
PubMed1990

The malignant conversion step of mouse skin carcinogenesis.

Multiple benign squamous papillomas commonly precede the development of an occasional squamous cell carcinoma in mouse skin carcinogenesis. The incidence of carcinomas can be enhanced by treating papilloma-bearing mice with mutagens such as urethane, nitroquinoline-N-oxide, or cisplatinum. This observation suggests that a genetic change is required for malignant conversion. The malignant phenotype is characterized by a marked reduction in the transcription of specific epidermal differentiation markers, a pattern which is useful for the early diagnosis of malignant conversion. Cells expressing a benign phenotype can be obtained by introducing the v-rasHa oncogene into cultured epidermal cells by a replication-defective retrovirus. Alternatively, benign tumor cells can be cultured from papillomas induced by chemical carcinogens in vivo or from carcinogen-treated mouse epidermis. In all cases, the benign phenotype in vitro is characterized by an altered biological response to changes in extracellular calcium, an important determinant of the differentiation state of cultured normal keratinocytes. Transfection of cloned plasmid DNA into benign tumor cells has revealed that transforming constructs of the fos oncogene induce malignant conversion, whereas myc and adenovirus E1A oncogenes do not. The fos carcinomas do not express differentiation-specific epidermal markers and secrete proteases such as transin and urokinase, a set of characteristics previously noted for chemically induced skin carcinomas. Cultured normal epidermal cells, exposed to the v-ras and the v-fos oncogenes simultaneously, are malignantly transformed. Alone, the fos oncogene does not detectably alter the phenotype of normal keratinocytes. These studies indicate that a limited number of genes is involved in epidermal carcinogenesis.

Animals↗
PubMed1990

Keratins 1 and 10 or homologues as regular constituents of inner root sheath and cuticle cells in the human hair follicle.

Morphological maturation of the inner root sheath (IRS) and cuticle of the human hair follicle reveals analogies to differentiation processes in other keratinizing epithelia. Detailed biochemical analysis of respective differentiation products, however, has been largely restricted by their low solubility. Herein we provide further evidence for the existence of K1 and K10-derivatives in IRS and hair cuticle based on protein analysis of isolated fractions and immunofluorescence in situ, substantiating our earlier data (Stark, H. J., et al. Differentiation 35, 236-248 (1987)). Extracts from both compartments showed on two-dimensional (2D)-polyacrylamide gels a group of presumptive K1 and K10-turnover products in a wide pI (basic to acidic) and Mr range (56,000-65,000), named IC-I to III and IC-IV, respectively. These components (also found in nail plate) reacted with specific antibodies (to K1 and K10) on Western blots. Weak but distinctive radiolabeling of presumptive precursor spots close to authentic K1 and K10, respectively, and their presence in lower follicle fractions (distant from infundibulum) largely precluded epidermal contamination. Two-dimensional tryptic peptide maps of excised 2D spots from the IC-I to III series revealed high homology to K1, and those from IC-IV components to K10. Immunodetection in frozen sections was improved by trypsin pretreatment and showed distinguished staining for K1 and K10 in IRS ranging from the lower bulbus region up to the "keratinizing zone" of the follicle. Above, the reaction was abruptly abolished which coincides with ultrastructural "melting" of distinct filaments in the intracellular matrix. Thus, our data suggest that differentiation in these follicular compartments (IRS and cuticle) might follow common principles of keratinization.

Fluorescent Antibody Technique↗
PubMed1989

Density-dependent modulation of synthesis of keratins 1 and 10 in the human keratinocyte line HACAT and in ras-transfected tumorigenic clones.

The spontaneous human keratinocyte line HaCaT and c-Ha-ras oncogene-transfected cell clones are capable of expressing an unusually broad spectrum of keratins, not observed so far in epithelial cells. This expression is, however, strongly modulated by environmental conditions, including cell density. Both cells of the nontumorigenic HaCaT line and the tumorigenic HaCaT-ras clones, I-7 and II-3 (giving rise to benign and malignant tumors, respectively), constitutively expressed the keratins K5, K6, K14, K16 and K17, which are also common in cultures of normal keratinocytes. In addition keratins K7, K8, K18 and K19, generally associated with simple epithelia, were synthesized (to a most pronounced extent in sparse cultures), while keratins K4, K13 and K15 appeared at confluence, presumably with the onset of stratification. Moreover, in both HaCaT and HaCaT-ras clones the epidermal "suprabasal" keratins, K1 and K10, were expressed in conventional submerged cultures (at normal vitamin A levels), markedly rising with cell density, but not strictly correlated with the degree of stratification. This property was maintained in HaCaT cells up to the highest passages. According to immunofluorescence, this was due to increasing numbers of strongly stained cells, and not due to a gradual increase in all cells. Most strikingly, there was a significant delay in the appearance of K10 compared to K1, and this dissociation of expression was most evident in dispase-detached cell sheets (submerged cultures) and organotypic cultures of the ras clones (grown at the air-liquid interface). While on frozen sections bright staining for K1 was seen in some basal and virtually all suprabasal cell layers, K10 was largely restricted to the uppermost layers. Thus, obviously synthesis of K1 and K10 can be regulated independently, although generally in this given sequence. The apparent compatibility of K1 synthesis with proliferation and particularly the extended delay of K10 expression (as a postmitotic event) might be causally related to altered growth control and as such imply the significance of this disturbance. Finally, the highly preserved epidermal characteristics, in terms of expression of keratins (and other differentiation markers [5]) and their regulation, makes these cell lines excellent candidates for studying external modulators of differentiation and also underlying molecular mechanisms.

Antibody Specificity↗
PubMed1988

Isolation of complementary DNA for bullous pemphigoid antigen by use of patients' autoantibodies.

Autoantibodies from bullous pemphigoid (BP) patients define a 230-kD protein found in the basement membrane of stratified squamous epithelia. The purpose of this study was to isolate and characterize a cDNA clone with coding sequences for BP antigen. Poly(A+) RNA derived from total RNA of cultured keratinocytes was used, with oligo-dT priming, to construct a cDNA library in the lambda gt11 expression vector, which was screened by the immunoperoxidase method with one BP serum. One darkly stained clone, called here the BP clone, was further characterized. 9 of 9 BP sera, but none of 6 normal and 11 pemphigus sera, bound the plaques of this BP clone. Furthermore, BP IgG affinity purified on plaques of this clone, but not unrelated clones, bound the epidermal basement membrane by immunofluorescence and immunoprecipitated the 230-kD BP antigen from extracts of cultured keratinocytes. Eco RI digestion of the BP clone's cDNA insert demonstrated a 680- and 1,500-bp fragment. Northern blots of total keratinocyte RNA showed that complementary riboprobes transcribed from both fragments hybridized to a 9-kb RNA. Dideoxy DNA sequencing from the 5' end of the BP cDNA demonstrated a 1,992-bp open reading frame, encoding a peptide of 76 kD. This BP cDNA clone will be valuable for understanding the protein structure, expression, and gene organization of BP antigen.

Amino Acid Sequence↗
PubMed1987

Cultivation of murine hair follicles as organoids in a collagen matrix.

Techniques are described for the isolation and cultivation of functionally intact mouse hair follicles. Follicles were isolated by collagenase digestion of dermis from 5-day-old mice and purified by differential centrifugation and filtration. Purified follicles were cultured in a Type 1 collagen matrix using Medium 199 and 8% fetal calf serum as the basic nutrient. Viability of follicles was maintained in culture since the cultures incorporated thymidine into DNA and methionine into proteins for at least 7 days. Furthermore, follicles isolated from the collagen matrix after 7 days could reattach to a plastic culture substrate or be further cultivated in a fresh collagen matrix. Functional integrity of cultured follicles was maintained since some follicle-specific cytoskeletal proteins were synthesized in vitro, and follicles isolated from the collagen matrix after 7 days formed a haired skin when recombined with dermal fibroblasts and grafted to a skin site on nude mice. Only a minority of follicles appeared to produce a mature hair shaft in vitro by morphologic criteria, however, and synthesis of the total complement of hair proteins was not observed. Cholera toxin was a strong mitogen for cultured follicles, whereas epidermal growth factor was slightly mitogenic. Epidermal growth factor stimulated the release of a Type 1 collagenase by follicle cells, however. This model system provides an opportunity for the systematic analysis of factors required for the induction of hair growth and the underlying physiology of hair follicle development. This model should also be useful for studying the role of the hair follicle in skin carcinogenesis.

Animals↗
PubMed1985

Vitamin A: a key nutrient for the maintenance of epithelial differentiation.

Vitamin A deficiency or benzo(a)pyrene instillation into tracheas of Syrian golden hamsters causes squamous metaplasia of tracheobronchial epithelium, normally a mucous secretory tissue. In the present studies, we have employed a tracheal organ culture system and have reproduced the in vivo phenomenon of squamous metaplasia during culturing under vitamin A free conditions as well as after carcinogen treatment. The squamous metaplasia induced by vitamin A deficiency, both in vivo and in vitro, was accompanied by an overall increase in keratin synthesis. Vitamin A deficient tracheas were shown to contain keratins of 50, 48, 46.5 Kd detected with the antibody AE1, and 58, 56 and 52 Kd detected with AE3. These proteins were either absent or present in much less quantity in control tracheas. In deficient tracheas 60 kd keratin was found to be located specifically in squamous suprabasal cells, and 55 and 50 Kd keratin proteins were found in a greatly expanded basal cell compartment. Following carcinogen exposure, the appearance of 60 kd keratin and the enhanced expression of 50 and 55 Kd keratins preceded the squamoid metaplastic response as detected morphologically. Both the keratin changes and the morphological changes were prevented by retinoid treatment.

Animals↗