Search PubMed⌕ Search

Biomedical subjects

L B Taichman

Publications and source records attributed to L B Taichman.

At least 55 records · Page 3Linked to original sources
PubMed1989

Systemic distribution of apolipoprotein E secreted by grafts of epidermal keratinocytes: implications for epidermal function and gene therapy.

In the present study, human apolipoprotein E (apoE) was monitored in the circulation of athymic mice and rats bearing human epidermal grafts. Human apoE was detected in the systemic circulation of graft-bearing animals as long as the graft remained on the animal. Within 24 hr of graft removal, human apoE was not detectable in plasma, indicating that apoE resulted from continuous production of the protein by grafted keratinocytes. These results show that proteins as large as apoE (299 amino acids) traverse the epidermal-dermal barrier and achieve systemic distribution where they may produce effects on distal tissues. The feasibility of using grafts of genetically-altered keratinocytes for the delivery of secreted proteins is clearly reinforced by the demonstration that an epidermally derived protein exhibits systemic distribution. Finally, by virtue of its systemic distribution, apoE produced in a peripheral tissue such as skin, may function in the reverse transport of cholesterol from peripheral tissues to the liver.

Animals↗
PubMed1989

Transient expression of a transfected gene in cultured epidermal keratinocytes: implications for future studies.

We examine the effect of keratinocyte differentiation upon transient expression of a nonepithelial gene following DNA-mediated transfer. Cultures of primary epidermal keratinocytes were transfected with the reporter gene, chloramphenicol acetyltransferase (CAT). The CAT gene was linked at the 5' end to the long terminal repeat (LTR) regulatory sequences from Rous sarcoma virus, and gene transfer was accomplished by the calcium phosphate coprecipitation method. Transfected cells were fractionated on Ficoll 400 density gradients. The major finding of this study was that the larger, more differentiated cells displayed five- to seven-fold higher levels of CAT activity per cell than the smaller, less differentiated cells. The higher levels of CAT activity did not result from greater uptake of DNA because cells of all gradient fractions contained one to two copies of plasmid DNA per cell. Furthermore, the CAT gene linked to the regulatory sequences from another virus, SV40, gave the same result. We conclude that the CAT gene, when controlled by these viral regulatory sequences, is expressed more efficiently in differentiated keratinocytes. These results have important implications for the interpretation of future studies of gene expression in transfected keratinocytes.

Avian Sarcoma Viruses↗
PubMed1989

Synthesis and secretion of apolipoprotein E by cultured human keratinocytes.

Non-polar lipids are synthesized by keratinocytes in the epidermis and transported to the extracellular space where they contribute to formation of a permeability barrier. Transport of non-polar lipids in other organs and tissues usually occurs with the lipid complexed to an apolipoprotein. In this study we set out to learn if apolipoprotein E is produced by human epidermal keratinocytes in culture. Analysis of total cellular RNA from cultured keratinocytes showed the presence of human apolipoprotein E mRNA at concentrations ranging from 2.5 to 35 molecules/cell. The cells secrete a protein identified as apo E on the basis of molecular weight, isoform pattern, and immunoreactivity. Enzyme linked immunosorbent assay of media from keratinocyte cultures indicated that apolipoprotein E is secreted at a rate of 0.92 ng/h/10(6) cells.

Apolipoproteins E↗
PubMed1989

Tissue site-specific enhancer function of the upstream regulatory region of human papillomavirus type 11 in cultured keratinocytes.

The human papillomavirus type 11 regulatory region was cloned upstream of a reporter complex and microinjected into nuclei of individual primary human keratinocytes. Genital and laryngeal keratinocytes, normal host cells for this virus, exhibited higher levels of expression than cutaneous keratinocytes. We conclude that a papillomavirus enhancer(s) shows preferences among epithelial cells from different tissue sites.

Antigens, Viral, Tumor↗
PubMed1988

The effect of growth-promoting agents on replication and cell cycle withdrawal in cultures of epidermal keratinocytes.

Epidermal keratinocytes grow in culture to form a stratified squamous epithelium. These cultures contain a replicating as well as a terminally differentiating population and undergo surface desquamation. Epidermal growth factor (EGF) and cholera toxin are usually employed as growth-promoting agents because they reduce the population doubling time; that is, the period required to increase the total cell number twofold. There are three ways in which this reduction in population doubling time could be achieved: (a) the time for one cell cycle or the cell cycle length may be shortened; (b) the number of cells that withdraw from the cell cycle and terminally differentiate may be reduced; or (c) the number of cells that desquamate into the medium over a set period of time may be reduced. We have explored these possibilities in growing cultures of epidermal keratinocytes using a newly developed double-label assay. This assay gives a measure of both cell length and cell cycle withdrawal. Results show that the growth enhancement induced by EGF and cholera toxin can be attributed primarily to a reduction in cell cycle withdrawal and, to a lesser degree, to a reduction in cell cycle length. EGF and cholera toxin have no significant effect on the rate of desquamation. A linear correlation was noted between cell cycle lengths and withdrawal, suggesting an interconnection between the rate of cell renewal and the likelihood of undergoing terminal differentiation.

Cell Cycle↗
PubMed1988

Epithelium-specific response of cultured keratinocytes to infection with adenovirus type 2.

Adenoviruses are pathogenic for certain stratified squamous epithelia. The sites most frequently involved are the upper respiratory tract and oropharynx. Adenovirus infections of the epidermis are quite rare. We examined the virus-cell interactions of adenovirus type 2 (Ad2) and cultured human keratinocytes grown from a variety of body sites. Our intent was to explore the nature of the apparent epithelium-specific susceptibility to Ad2. In brief, we found that in vitro viral susceptibility of the keratinocytes could be reliably predicted based on whether the cells originated from an epidermal or oropharyngeal surface. Ad2 proceeded through a complete vegetative cycle when used to infect cultured keratinocytes from oropharyngeal sites (e.g., gingiva and soft palate). In contrast, Ad2 infection was severely restricted in keratinocytes from epidermal sites (e.g., foreskin, abdomen, and buttock). These results demonstrate that the in vitro response to infection with Ad2 reflects in vivo tissue-specific susceptibility. In vivo, cervical epithelium is rarely infected with Ad2 and yet in culture, cervical keratinocytes were fully permissive for Ad2 replication. We propose that the permissive or nonpermissive response to Ad2 may be regulated by a particular aspect of cell phenotype. Because the permissive responses seen in this study were all generated in keratinocytes from mucosal sites, it is possible the in vitro response to Ad2 reflects inherent differences between mucosal and epidermal keratinocytes.

Adenoviruses, Human↗
PubMed1988

Evidence for two points of restriction in the expression of adenovirus type 2 in cultured epidermal keratinocytes.

Cultures of epidermal keratinocytes contain two populations of cells, a basal undifferentiated population and a suprabasal terminally differentiated population. When exposed to wild-type adenovirus type 2 (wtAd2), the suprabasal cells are positive by immunofluorescence for capsid antigen and exhibit cytopathic effects (CPE) (R.F. LaPorta, and L.B. Taichman, Virology 110:137-146, 1981). The basal cells, although infected, are not positive for capsid antigen and do not display CPE. Despite CPE and capsid antigens in suprabasal cells, yields of virus from the entire culture are very low (10 PFU per cell). These observations suggest that Ad2 expression is restricted at different times in the viral life cycle in basal and suprabasal cells. To test this hypothesis, we isolated host range (hr) mutants of Ad2 on two lines of squamous cell carcinoma (SCC) keratinocytes which were shown to be restrictive for wtAd2 replication. The hrAd2 mutants produced high yields of progeny virus in epidermal cell cultures (500 to 600 PFU per cell). However, the pattern of CPE induction in these cultures was like that produced by wtAd2, i.e., basal cells were CPE negative and suprabasal cells were CPE positive. The high yield of hrAd2 progeny indicated that the restriction present in suprabasal cells was overcome. However, the failure of hrAd2 mutants to induce CPE in basal cells indicated that the hrAd2 mutants remain restricted in the basal population and supported our hypothesis that a second and distinct restriction exists in basal keratinocytes.

Adenoviridae↗
PubMed1987

Cell-cycle withdrawal in cultured keratinocytes.

Cell-cycle withdrawal is the irreversible arrest of replication that occurs in keratinocytes early in terminal differentiation. According to the epidermal proliferation unit (EPU) model of renewal, withdrawal takes place in a subset of cells that have completed a final cycle of amplification replication. Using a recently developed double-labelling assay, we followed cell-cycle withdrawal in growing cultures of epidermal keratinocytes and correlated these results with population-growth kinetics. The levels of withdrawal measured were much too high to be consistent with the population-growth kinetics. These unexpectedly high levels could be explained by postulating that withdrawal takes place in a specific subset of cells, as described by the EPU model. Other possible explanations were entertained but, for various reasons, were considered unlikely. To learn whether withdrawal occurred in basal or suprabasal cells, confluent cultures were pulse labeled with 3H-thymidine, and the position of the labeled cells was monitored by autoradiography during the period of cold chase. The results indicated that, in cultured keratinocytes, withdrawal takes place while the cells are still in the basal compartment.

Bromodeoxyuridine↗
PubMed1987

Identification and mapping of human papillomavirus type 1 RNA transcripts recovered from plantar warts and infected epithelial cell cultures.

Multiple spliced transcripts of human papillomavirus type 1 were detected by electron microscopic analysis of R-loops formed with total RNA extracted from plantar warts and with poly(A)+ RNA isolated from cultured keratinocytes infected with human papillomavirus type 1. The 5' ends of the RNAs were mapped to sites in the E7 open reading frame (ORF), just upstream of the E6 ORF and in the upstream regulatory region. Species with 5' ends in E7 accounted for over 95% of all transcripts seen. Two polyadenylation sites were used, one at the end of the early (E) region of the viral DNA, the other at the end of the late (L) region. The most abundant species had a short 5' exon of approximately 100 nucleotides spanning the junction of the E7 and E1 ORFs spliced to a 3' exon of 800 nucleotides in the region with overlapping E2 and E4 ORFs; it was polyadenylated at the end of the E region. This species probably encodes the abundant E4 protein found in plantar warts (F. Breitburd, O. Croissant, and G. Orth, Cancer Cells, vol. 5, in press; J. Doorbar, D. Campbell, R. J. A. Grand, and P. H. Gallimore, EMBO J. 5:355-362, 1986). Other transcripts had exons spanning the E6-E7 ORFs, the E4-E5-L2-L1 ORFs, or the L1 ORF. The infrequent L1 transcript, probably the mRNA coding for the major capsid protein, had the same 5' exon in E7 as the abundant mRNA spliced from E1 and E4 ORFs, suggesting genetic regulation via the choice of the alternative polyadenylation sites or mRNA processing.

Base Sequence↗
PubMed1986

Heterogeneity in the replicating population of cultured human epidermal keratinocytes.

We studied the replication of keratinocytes in stratified squamous epithelia. Other studies have revealed functional and morphological heterogeneity in the replicating population of such cells. To examine possible kinetic heterogeneity, we determined the cell-cycle lengths of replicating cells in cultures of human epidermal keratinocytes. A double-label assay was developed, which measures the time between two successive cycles of DNA synthesis. The first cycle of DNA synthesis was marked by pulse labeling cultures for a brief period with 14C-thymidine (dThd), and the second cycle was detected by labeling at a later time with bromodeoxyuridine (BrdUrd). The time taken for the 14C-labeled DNA to become doubly labeled with BrdUrd was shown to correspond to the length of the cell cycle. In subconfluent cultures in which the cell number increased at an exponential rate, the average cell-cycle time was 21.5 h. In confluent cultures in which desquamation was balanced by cell renewal, the average cell cycle was 31.5 h. However, in confluent cultures, three populations of replicating cells were evident, these having cycle times of 22, 33, and 40 h. In subconfluent cultures, there was no clear evidence for cell-cycle heterogeneity of the replicating cells, although the most rapidly cycling cells in these cultures had a cycle time (16 h) considerably less than the most rapidly cycling cells in the confluent cultures (21 h). It is possible that the rapidly cycling cells seen in the subconfluent cultures were stem cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Cycle↗
PubMed1984

Kinetics of withdrawal from the cell cycle in cultured human epidermal keratinocytes.

In keratinizing epithelia one of the earliest changes in the process of terminal differentiation is cessation of replication or withdrawal from the cell cycle. In this report, we measured the loss of colony-forming ability, and confirmed that withdrawal from the cell cycle is a specific event that occurs during maturation of the keratinocyte in culture. In addition, the rate of withdrawal was assayed by labeling cultures for 24 h with [14C]dThd and then measuring the fraction of labeled cells that undergo repeated cycles of DNA synthesis. These additional cycles of replication were measured by feeding BrdUrd to the cultures and quantitating the distribution of 14C-labeled DNA in unsubstituted and BrdUrd-substituted DNA in CsCl density gradients. The results show that the fraction of 14C-labeled DNA undergoing replication decreases exponentially by 23% every 24 h. This cessation of replication could not be explained by a reduced level of replication in the entire culture since during each day of the experiment about 8% of the total DNA underwent replication. The exponential decrease in replication of 14C-labeled DNA represents withdrawal from the cell cycle. Since the doubling time for keratinocytes is approximately 24 h, these results suggest that following each cycle of replication, there is a probability of 0.23 that postreplicated cells will withdraw from the cell cycle.

Cell Cycle↗
PubMed1984

The search for a culture system for papillomavirus.

Papillomaviruses induce tumors of keratinocytes. Vegetative viral DNA replication and virion assembly are seen in those cells which are in the process of keratinizing or are keratinized. To date, no cell culture system has been developed that permits expression of the complete viral life cycle. Keratinocytes infected in culture may harbor the virus as a stable, replicating episome, but they do not support vegetative viral growth, nor do they become immortalized or transformed. The major obstacle in using keratinocyte cultures may be related to a dual need for transformation and full differentiation. Some animal papillomaviruses have been shown to be capable of transforming cultured murine fibroblasts. The fibroblast model is useful for identifying the viral-transforming gene(s) and their products.

Animals↗
PubMed1983

The role of keratinocyte differentiation in the expression of epitheliotropic viruses.

We have examined the growth of three epitheliotropic viruses in cultures of human epidermal keratinocytes: herpes simplex virus (HSV) type 1, adenovirus type 2 (Ad-2), and human papillomavirus (HPV) type 1. Differences were noted in the level of expression of each virus, and these differences may be related to a dependency or lack of dependency on keratinocyte differentiation for complete viral growth. Of the three viruses studied, HSV was the only one to replicate productively in all cells of the culture. Its expression was independent of keratinocyte differentiation. This is unlike Ad-2, which infected all cells in the culture but replicated productively only in the suprabasal cells. Basal keratinocytes were shown to be infected, but for unknown reasons, they appeared in most instances to be nonpermissive for Ad-2 replication. Infected basal keratinocytes became permissive when they reached a suprabasal position. Ad-2 appears to require keratinocyte differentiation for full expression in culture. Following infection with HPV, cultured keratinocytes showed no evidence of productive replication. However, 50 to 250 copies of HPV DNA could be detected in each cell (average) as stable nonintegrated molecules. Viral DNA replication has been shown to occur in the younger cells and not in the older, more differentiated keratinocytes. The failure of HPV to be fully expressed in culture may be related, in part, to incomplete differentiation of the keratinocyte in vitro. The major conclusions of this study are (1) that keratinocyte differentiation is likely to play a role in the expression of some epitheliotropic viruses in culture, and (2) that keratinocyte differentiation may be a factor in the pathogenesis of certain viral diseases of keratinizing epithelia.

Adenoviridae↗
PubMed1982

Human papilloma viral DNA replicates as a stable episome in cultured epidermal keratinocytes.

Human papilloma virus (HPV) is poorly understood because systems for its growth in tissue culture have not been developed. We report here that cultured human epidermal keratinocytes could be infected with HPV from plantar warts and that the viral DNA persisted and replicated as a stable episome. There were 50-200 copies of viral DNA per cell and there was no evidence to indicate integration of viral DNA into the cellular genome. There was also no evidence to suggest that viral DNA underwent productive replication. We conclude that cultured human epidermal keratinocytes may be a model for the study of certain aspects of HPV biology.

Cell Division↗
PubMed1982

Synthesis of keratin proteins during maturation of cultured human keratinocytes.

We have examined the synthesis of keratin and total protein during cellular maturation in cultured oral and epidermal keratinocytes. Keratinocytes were separated on the basis of maturational age on gradients of Ficoll 400. Analysis of selected gradient fractions revealed that during maturation in culture the amount of protein/cell increases from 100 to 900 pg and the apparent rate of protein synthesis increases about two-fold. However, the proportion of protein that is keratin remains constant throughout this process. The major keratin proteins (58K, 56K, and 46K) are synthesized at the same relative rates throughout maturation in vitro. Therefore, during terminal differentiation in culture, the keratinocyte forms an expanding cell in which the rate of protein synthesis increases while the relative proportion of keratin proteins and nonkeratin proteins remain the same.

Cell Survival↗