Lorne Taichman - Academia.edu (original) (raw)
Papers by Lorne Taichman
Biochemistry, May 3, 1977
The distribution of chromatin-associated proteins in replicating Chinese Hamster ovary cells has ... more The distribution of chromatin-associated proteins in replicating Chinese Hamster ovary cells has been examined using the method described by Taichman and Freedlender (Taichman, L., and Freedlender, E. F. (1976), Biochemistry 15, 447). Cells are grown for several generations in [I4C]lysine and thymidine, and then for one generation in the presence of [)HI lysine and 5-bromodeoxyuridine (BrUdRib) and a further generation in cold amino acid and BrUdRib. This protocol produces equal amounts of unifilarly (heavy-light) and bifilarly (heavy-heavy) substituted DNA. Chromatin containing the two types of DNA are separated by sucrose-gradient cen-M a n y types of eukaryotic cells divide to produce daughter cells identical to the parent. Although the molecular systems controlling gene expression are maintained during replication, the details of this process are not understood. The possibility that control might be achieved by using a self-perpetuating arrangement of proteins on D N A was briefly considered by one of us some time ago (Smithies, 1970). Control proteins associated with D N A in a differentiated cell were postulated to direct the assembly of newly synthesized proteins onto newly replicated DNA to form DNA-protein complexes identical to those initially present. Tsanev and Sendov (1971) considered a similar situation in much greater detail when they postulated that the parental histones remain associated with the parental strand of DNA and direct the association of newly synthesized histones with the newly synthesized strand of DNA. Such nonrandom distribution, referred to as segregation, is the subject of this investigation. The question of how parental chromosomal proteins are distributed to daughter chromatin has been asked previously but a clear case for either the random or the nonrandom mode has not been obtained. Prescott and Bender (1963) used autoradiography to follow the fate of pulse-labeled proteins in Chinese Hamster cells in tissue culture. They found little conservation of the labeled proteins; the small amount of label that was retained was equally distributed to both chromatids, indicating random association. More recently, Tsanev and Russev (1 974) have obtained evidence in regenerating rat liver suggesting that some newly synthesized chromosomal proteins preferentially associate with newly synthesized D N A as predicted by the segregation model. While the proteins were re
Gene Therapy, Oct 1, 1997
There is now strong evidence that the chorioretinal ornithine disappearance from the medium that ... more There is now strong evidence that the chorioretinal ornithine disappearance from the medium that is signifidegeneration associated with ornithine-␦-aminotransferase cantly higher than the rate of disappearance from the (OAT) deficiency is a consequence of hyperornithinemia. medium bathing normal keratinocytes. In addition, OAT Therefore development of a metabolic system for clearing activity determined in soluble protein prepared from sonornithine from the circulation is being pursued as a poten-icates suggests that the capacity to maintain plasma ornitial treatment. The skin is considered an attractive location thine within the normal range is contained within an for such a metabolic system because autologous cells can accomplishable graft of keratinocytes overexpressing OAT. be safely and easily utilized. This study was undertaken to However, the actual rate of ornithine disappearance from determine the ornithine metabolizing capacity of epidermal the media was significantly less than predicted from keratinocytes expressing normal and superphysiologic enzyme activity assays. Following ornithine metabolite proamounts of OAT. The data show that overexpression of duction by intact cells suggests that ornithine metabolism OAT in keratinocytes cultured from a gyrate atrophy patient is limited primarily by clearance of downstream metabrestores ornithine metabolism and results in a rate of olites, as opposed to substrate delivery.
Cold Spring Harbor Symposia on Quantitative Biology, 1978
Experimental Dermatology, Oct 1, 1999
Recent progress with innovative, experimental gene therapy approaches in animals, and recent impr... more Recent progress with innovative, experimental gene therapy approaches in animals, and recent improvements in our understanding and manipulation of stem cells, gene expression and gene delivery systems, have raised plenty of hopes in essentially all branches of clinical medicine that hitherto untreatable or poorly manageable diseases will soon become amenable to treatment. Few other organ systems have received such enthusiastic reviews in recent years as to the chances and prospects of gene therapy as the skin, with its excellent accessibility and its pools of-seemingly-readily manipulated epithelial stem cells (cf.
Human Gene Therapy, Nov 20, 1997
Gyrate atrophy is a progressive blindness associated with deficiency of ornithine aminotransferas... more Gyrate atrophy is a progressive blindness associated with deficiency of ornithine aminotransferase (OAT). The strategy of using an autologous keratinocyte graft, modified to express high levels of OAT as an ornithine-catabolizing skin-based enzyme sink, is investigated. Two OAT-containing retroviral vectors were constructed with or without a resistance gene. When packaged in a retroviral vector particle generated with the gibbon ape leukemia (GALV) virus envelope (PG13), these vectors could readily transduce >50% of target keratinocytes. The transduced keratinocytes in culture expressed up to 75-fold more OAT than normal control keratinocytes and these gene-modified cells extracted [14C]ornithine more efficiently than controls. The vector prepared without neo transduced cells more efficiently and led to higher levels of OAT expression than the neo-containing vector. Ornithine catabolism was maintained at high levels when the transduced patient keratinocytes were differentiated in vitro as a multilayered cutaneous organoid.
Archives of Oral Biology, 1982
keratinocytes from epidermis, gingiva and buccal mucosa are cultured in vitro they form a stratif... more keratinocytes from epidermis, gingiva and buccal mucosa are cultured in vitro they form a stratified squamous epithelium that lacks evidence of orthokeratinization or parakeratinization. We attempted to induce orthokeratinization or parakeratinization in cultured gingival keratinocytes by co-cultivation with fibroblasts from human skin, gingiva and buccal rnucosa. Keratinization was defined by the morphological appearance of the cultured cells and by the presence of large molecular weight keratin proteins (63,000 and 67,000 mol. wt). Using these criteria, the chosen fibroblasts failed to induce any alteration in the pattern of keratinization. We conclude that under our present culture conditions, fibroblasts alone cannot induce keratinization in cultured keratinocytes.
Journal of Virology, Nov 1, 1988
Journal of Virology, 1988
Cultures of epidermal keratinocytes contain two populations of cells, a basal undifferentiated po... more 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 epiderm...
Journal of Investigative Dermatology, 1988
Human Gene Therapy, 1997
Gyrate atrophy is a progressive blindness associated with deficiency of ornithine aminotransferas... more Gyrate atrophy is a progressive blindness associated with deficiency of ornithine aminotransferase (OAT). The strategy of using an autologous keratinocyte graft, modified to express high levels of OAT as an ornithine-catabolizing skin-based enzyme sink, is investigated. Two OAT-containing retroviral vectors were constructed with or without a resistance gene. When packaged in a retroviral vector particle generated with the gibbon ape leukemia (GALV) virus envelope (PG13), these vectors could readily transduce >50% of target keratinocytes. The transduced keratinocytes in culture expressed up to 75-fold more OAT than normal control keratinocytes and these gene-modified cells extracted [14C]ornithine more efficiently than controls. The vector prepared without neo transduced cells more efficiently and led to higher levels of OAT expression than the neo-containing vector. Ornithine catabolism was maintained at high levels when the transduced patient keratinocytes were differentiated in vitro as a multilayered cutaneous organoid.
Experimental Dermatology, 1999
Recent progress with innovative, experimental gene therapy approaches in animals, and recent impr... more Recent progress with innovative, experimental gene therapy approaches in animals, and recent improvements in our understanding and manipulation of stem cells, gene expression and gene delivery systems, have raised plenty of hopes in essentially all branches of clinical medicine that hitherto untreatable or poorly manageable diseases will soon become amenable to treatment. Few other organ systems have received such enthusiastic reviews in recent years as to the chances and prospects of gene therapy as the skin, with its excellent accessibility and its pools of – seemingly – readily manipulated epithelial stem cells (cf. Cotsarelis et al., Exp Dermatol 1999: 8: 80–88).However, as in other sectors of clinical medicine, the actual implementation of general gene therapy strategies in clinical practice has been faced with a range of serious difficulties (cf. Smith, Lancet 1999: 354 (suppl 1): 1–4; Lattime & Gerson (eds.), Gene Therapy of Cancer, Academic Press, San Diego, 1999). Thus, it ...
Biochemistry, 1977
The distribution of chromatin-associated proteins in replicating Chinese Hamster ovary cells has ... more The distribution of chromatin-associated proteins in replicating Chinese Hamster ovary cells has been examined using the method described by Taichman and Freedlender (Taichman, L., and Freedlender, E. F. (1976), Biochemistry 15, 447). Cells are grown for several generations in [I4C]lysine and thymidine, and then for one generation in the presence of [)HI lysine and 5-bromodeoxyuridine (BrUdRib) and a further generation in cold amino acid and BrUdRib. This protocol produces equal amounts of unifilarly (heavy-light) and bifilarly (heavy-heavy) substituted DNA. Chromatin containing the two types of DNA are separated by sucrose-gradient cen-M a n y types of eukaryotic cells divide to produce daughter cells identical to the parent. Although the molecular systems controlling gene expression are maintained during replication, the details of this process are not understood. The possibility that control might be achieved by using a self-perpetuating arrangement of proteins on D N A was briefly considered by one of us some time ago (Smithies, 1970). Control proteins associated with D N A in a differentiated cell were postulated to direct the assembly of newly synthesized proteins onto newly replicated DNA to form DNA-protein complexes identical to those initially present. Tsanev and Sendov (1971) considered a similar situation in much greater detail when they postulated that the parental histones remain associated with the parental strand of DNA and direct the association of newly synthesized histones with the newly synthesized strand of DNA. Such nonrandom distribution, referred to as segregation, is the subject of this investigation. The question of how parental chromosomal proteins are distributed to daughter chromatin has been asked previously but a clear case for either the random or the nonrandom mode has not been obtained. Prescott and Bender (1963) used autoradiography to follow the fate of pulse-labeled proteins in Chinese Hamster cells in tissue culture. They found little conservation of the labeled proteins; the small amount of label that was retained was equally distributed to both chromatids, indicating random association. More recently, Tsanev and Russev (1 974) have obtained evidence in regenerating rat liver suggesting that some newly synthesized chromosomal proteins preferentially associate with newly synthesized D N A as predicted by the segregation model. While the proteins were re
Archives of Oral Biology, 1982
Journal of Investigative Dermatology, 1996
Journal of Investigative Dermatology, 1994
Recently it has been shown that apolipoprotein E (apoE) secreted by keratinocytes in transplanted... more Recently it has been shown that apolipoprotein E (apoE) secreted by keratinocytes in transplanted epidermal grafts reaches the systemic circulation. In this study we ask which cells in cultures of epidermal keratinocytes, basal or suprabasal, are the source of apoE. By fractionating disaggregated cultures in gradients of Fico1l400, the small nondifferentiated cell s derived from the basal compartment were shown to be the source of apoE. The larger more differentiated cells derived from supra basal layers could not be shown to contain or secrete apoE, although they did contain the apoE mRNA.
Human Gene Therapy, 1994
Grafts of autologous keratinocytes genetically altered to secrete a new gene product are a potent... more Grafts of autologous keratinocytes genetically altered to secrete a new gene product are a potential vehicle for gene therapy. To consider the feasibility of such an approach, we have examined the ability of keratinocytes to secrete and deliver apolipoprotein E (apoE) to the circulation of mice bearing grafts of human keratinocytes. The grafted keratinocytes secreted two forms of apoE, an endogenous apoE encoded in the genome and a recombinant apoE encoded in a transfected gene construct. In vitro studies showed that endogenous apoE was secreted from basal keratinocytes whereas recombinant apoE was secreted from basal as well as suprabasal cells. On the basis of amounts of recombinant apoE present in the serum of grafted mice, we estimate that a graft occupying 2% of the surface area of an adult human would deliver 6.5-8.3 mg of recombinant apoE protein per day.
Proceedings of the National Academy of Sciences, 1998
Epidermis is renewed by a population of stem cells that have been defined in vivo by slow turnove... more Epidermis is renewed by a population of stem cells that have been defined in vivo by slow turnover, label retention, position in the epidermis, and enrichment in β 1 integrin, and in vitro by clonogenic growth, prolonged serial passage, and rapid adherence to extracellular matrix. The goal of this study is to determine whether clonogenic cells with long-term growth potential in vitro persist in vivo and give rise to a fully differentiated epidermis. Human keratinocytes were genetically labeled in culture by transduction with a retrovirus encoding the lacZ gene and grafted to athymic mice. Analysis of the cultures before grafting showed that 21.1–27.8% of clonogenic cells with the capacity for >30 generations were successfully transduced. In vivo , β-galactosidase (β-gal) positive cells participated in the formation of a fully differentiated epithelium and were detected throughout the 40-week postgraft period, initially as loosely scattered clusters and later as distinct vertical ...
DNA Damage and Repair in Human Tissues, 1990
There is considerable excitement about the possibility of somatic cell gene therapy, that is, the... more There is considerable excitement about the possibility of somatic cell gene therapy, that is, the introduction and expression of defined genes into cells for the purpose of providing a needed gene product. Most current research on gene therapy has been focused on the use of marrow stem cells as a therapeutic vehicle (Belmont et al., 1986). Inherited hematological disorders, such as severe combined immunodeficiency caused by adenosine deaminase deficiency, are diseases that might be amenable to this form of therapy (Kellems et al., 1985). A possible approach would be through genetic transfer of a recombinant adenosine deaminase gene into autologous marrow stem cells, transplantation of the transformed cells into the patient, and establishment of a population of stem cells that give rise to immunologically competent lymphoid cells. Although many questions need to be answered before we know the efficacy of such therapy, the National Institute of Health has issued guidelines for its use, and clinical trials have already begun (Culliton, 1989).
Journal of Investigative Dermatology Symposium Proceedings, 2004
For long-term cutaneous gene therapy, the therapeutic gene must be targeted to stem cells and be ... more For long-term cutaneous gene therapy, the therapeutic gene must be targeted to stem cells and be stably transmitted to and expressed in descendant cells. Retroviral vectors are highly efficient in gene transfer to human keratinocyte stem cells in culture; however, they cannot transduce quiescent stem cells in vivo. As lentiviral vectors (LVV) transduce non-proliferating cells, their ability to target human epidermal stem cells was evaluated. LVV were highly efficient in gene transfer to clonogenic keratinocytes in vitro. Despite higher transgene DNA content and comparable levels of transgene mRNA, levels of transgene product directed by lentivectors were 3folds lower than that of retrovectors. When transduced keratinocytes were grafted onto mice, transgene expression persisted for at least 20 wk; however, transgene product was detected primarily in the uppermost layers of epidermis. Inclusion of an element that is known to facilitate nuclear export of intron-less transcripts, resulted in enhanced transgene expression in keratinocytes. In vivo transduction of xenografted human skin with these vectors resulted in efficient gene transfer to epidermal progenitor cells. These results demonstrate stem cell transduction by LVV and point out the utility of using these vectors for direct gene transfer to and sustained expression in human epidermis.
Biochemistry, May 3, 1977
The distribution of chromatin-associated proteins in replicating Chinese Hamster ovary cells has ... more The distribution of chromatin-associated proteins in replicating Chinese Hamster ovary cells has been examined using the method described by Taichman and Freedlender (Taichman, L., and Freedlender, E. F. (1976), Biochemistry 15, 447). Cells are grown for several generations in [I4C]lysine and thymidine, and then for one generation in the presence of [)HI lysine and 5-bromodeoxyuridine (BrUdRib) and a further generation in cold amino acid and BrUdRib. This protocol produces equal amounts of unifilarly (heavy-light) and bifilarly (heavy-heavy) substituted DNA. Chromatin containing the two types of DNA are separated by sucrose-gradient cen-M a n y types of eukaryotic cells divide to produce daughter cells identical to the parent. Although the molecular systems controlling gene expression are maintained during replication, the details of this process are not understood. The possibility that control might be achieved by using a self-perpetuating arrangement of proteins on D N A was briefly considered by one of us some time ago (Smithies, 1970). Control proteins associated with D N A in a differentiated cell were postulated to direct the assembly of newly synthesized proteins onto newly replicated DNA to form DNA-protein complexes identical to those initially present. Tsanev and Sendov (1971) considered a similar situation in much greater detail when they postulated that the parental histones remain associated with the parental strand of DNA and direct the association of newly synthesized histones with the newly synthesized strand of DNA. Such nonrandom distribution, referred to as segregation, is the subject of this investigation. The question of how parental chromosomal proteins are distributed to daughter chromatin has been asked previously but a clear case for either the random or the nonrandom mode has not been obtained. Prescott and Bender (1963) used autoradiography to follow the fate of pulse-labeled proteins in Chinese Hamster cells in tissue culture. They found little conservation of the labeled proteins; the small amount of label that was retained was equally distributed to both chromatids, indicating random association. More recently, Tsanev and Russev (1 974) have obtained evidence in regenerating rat liver suggesting that some newly synthesized chromosomal proteins preferentially associate with newly synthesized D N A as predicted by the segregation model. While the proteins were re
Gene Therapy, Oct 1, 1997
There is now strong evidence that the chorioretinal ornithine disappearance from the medium that ... more There is now strong evidence that the chorioretinal ornithine disappearance from the medium that is signifidegeneration associated with ornithine-␦-aminotransferase cantly higher than the rate of disappearance from the (OAT) deficiency is a consequence of hyperornithinemia. medium bathing normal keratinocytes. In addition, OAT Therefore development of a metabolic system for clearing activity determined in soluble protein prepared from sonornithine from the circulation is being pursued as a poten-icates suggests that the capacity to maintain plasma ornitial treatment. The skin is considered an attractive location thine within the normal range is contained within an for such a metabolic system because autologous cells can accomplishable graft of keratinocytes overexpressing OAT. be safely and easily utilized. This study was undertaken to However, the actual rate of ornithine disappearance from determine the ornithine metabolizing capacity of epidermal the media was significantly less than predicted from keratinocytes expressing normal and superphysiologic enzyme activity assays. Following ornithine metabolite proamounts of OAT. The data show that overexpression of duction by intact cells suggests that ornithine metabolism OAT in keratinocytes cultured from a gyrate atrophy patient is limited primarily by clearance of downstream metabrestores ornithine metabolism and results in a rate of olites, as opposed to substrate delivery.
Cold Spring Harbor Symposia on Quantitative Biology, 1978
Experimental Dermatology, Oct 1, 1999
Recent progress with innovative, experimental gene therapy approaches in animals, and recent impr... more Recent progress with innovative, experimental gene therapy approaches in animals, and recent improvements in our understanding and manipulation of stem cells, gene expression and gene delivery systems, have raised plenty of hopes in essentially all branches of clinical medicine that hitherto untreatable or poorly manageable diseases will soon become amenable to treatment. Few other organ systems have received such enthusiastic reviews in recent years as to the chances and prospects of gene therapy as the skin, with its excellent accessibility and its pools of-seemingly-readily manipulated epithelial stem cells (cf.
Human Gene Therapy, Nov 20, 1997
Gyrate atrophy is a progressive blindness associated with deficiency of ornithine aminotransferas... more Gyrate atrophy is a progressive blindness associated with deficiency of ornithine aminotransferase (OAT). The strategy of using an autologous keratinocyte graft, modified to express high levels of OAT as an ornithine-catabolizing skin-based enzyme sink, is investigated. Two OAT-containing retroviral vectors were constructed with or without a resistance gene. When packaged in a retroviral vector particle generated with the gibbon ape leukemia (GALV) virus envelope (PG13), these vectors could readily transduce >50% of target keratinocytes. The transduced keratinocytes in culture expressed up to 75-fold more OAT than normal control keratinocytes and these gene-modified cells extracted [14C]ornithine more efficiently than controls. The vector prepared without neo transduced cells more efficiently and led to higher levels of OAT expression than the neo-containing vector. Ornithine catabolism was maintained at high levels when the transduced patient keratinocytes were differentiated in vitro as a multilayered cutaneous organoid.
Archives of Oral Biology, 1982
keratinocytes from epidermis, gingiva and buccal mucosa are cultured in vitro they form a stratif... more keratinocytes from epidermis, gingiva and buccal mucosa are cultured in vitro they form a stratified squamous epithelium that lacks evidence of orthokeratinization or parakeratinization. We attempted to induce orthokeratinization or parakeratinization in cultured gingival keratinocytes by co-cultivation with fibroblasts from human skin, gingiva and buccal rnucosa. Keratinization was defined by the morphological appearance of the cultured cells and by the presence of large molecular weight keratin proteins (63,000 and 67,000 mol. wt). Using these criteria, the chosen fibroblasts failed to induce any alteration in the pattern of keratinization. We conclude that under our present culture conditions, fibroblasts alone cannot induce keratinization in cultured keratinocytes.
Journal of Virology, Nov 1, 1988
Journal of Virology, 1988
Cultures of epidermal keratinocytes contain two populations of cells, a basal undifferentiated po... more 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 epiderm...
Journal of Investigative Dermatology, 1988
Human Gene Therapy, 1997
Gyrate atrophy is a progressive blindness associated with deficiency of ornithine aminotransferas... more Gyrate atrophy is a progressive blindness associated with deficiency of ornithine aminotransferase (OAT). The strategy of using an autologous keratinocyte graft, modified to express high levels of OAT as an ornithine-catabolizing skin-based enzyme sink, is investigated. Two OAT-containing retroviral vectors were constructed with or without a resistance gene. When packaged in a retroviral vector particle generated with the gibbon ape leukemia (GALV) virus envelope (PG13), these vectors could readily transduce >50% of target keratinocytes. The transduced keratinocytes in culture expressed up to 75-fold more OAT than normal control keratinocytes and these gene-modified cells extracted [14C]ornithine more efficiently than controls. The vector prepared without neo transduced cells more efficiently and led to higher levels of OAT expression than the neo-containing vector. Ornithine catabolism was maintained at high levels when the transduced patient keratinocytes were differentiated in vitro as a multilayered cutaneous organoid.
Experimental Dermatology, 1999
Recent progress with innovative, experimental gene therapy approaches in animals, and recent impr... more Recent progress with innovative, experimental gene therapy approaches in animals, and recent improvements in our understanding and manipulation of stem cells, gene expression and gene delivery systems, have raised plenty of hopes in essentially all branches of clinical medicine that hitherto untreatable or poorly manageable diseases will soon become amenable to treatment. Few other organ systems have received such enthusiastic reviews in recent years as to the chances and prospects of gene therapy as the skin, with its excellent accessibility and its pools of – seemingly – readily manipulated epithelial stem cells (cf. Cotsarelis et al., Exp Dermatol 1999: 8: 80–88).However, as in other sectors of clinical medicine, the actual implementation of general gene therapy strategies in clinical practice has been faced with a range of serious difficulties (cf. Smith, Lancet 1999: 354 (suppl 1): 1–4; Lattime & Gerson (eds.), Gene Therapy of Cancer, Academic Press, San Diego, 1999). Thus, it ...
Biochemistry, 1977
The distribution of chromatin-associated proteins in replicating Chinese Hamster ovary cells has ... more The distribution of chromatin-associated proteins in replicating Chinese Hamster ovary cells has been examined using the method described by Taichman and Freedlender (Taichman, L., and Freedlender, E. F. (1976), Biochemistry 15, 447). Cells are grown for several generations in [I4C]lysine and thymidine, and then for one generation in the presence of [)HI lysine and 5-bromodeoxyuridine (BrUdRib) and a further generation in cold amino acid and BrUdRib. This protocol produces equal amounts of unifilarly (heavy-light) and bifilarly (heavy-heavy) substituted DNA. Chromatin containing the two types of DNA are separated by sucrose-gradient cen-M a n y types of eukaryotic cells divide to produce daughter cells identical to the parent. Although the molecular systems controlling gene expression are maintained during replication, the details of this process are not understood. The possibility that control might be achieved by using a self-perpetuating arrangement of proteins on D N A was briefly considered by one of us some time ago (Smithies, 1970). Control proteins associated with D N A in a differentiated cell were postulated to direct the assembly of newly synthesized proteins onto newly replicated DNA to form DNA-protein complexes identical to those initially present. Tsanev and Sendov (1971) considered a similar situation in much greater detail when they postulated that the parental histones remain associated with the parental strand of DNA and direct the association of newly synthesized histones with the newly synthesized strand of DNA. Such nonrandom distribution, referred to as segregation, is the subject of this investigation. The question of how parental chromosomal proteins are distributed to daughter chromatin has been asked previously but a clear case for either the random or the nonrandom mode has not been obtained. Prescott and Bender (1963) used autoradiography to follow the fate of pulse-labeled proteins in Chinese Hamster cells in tissue culture. They found little conservation of the labeled proteins; the small amount of label that was retained was equally distributed to both chromatids, indicating random association. More recently, Tsanev and Russev (1 974) have obtained evidence in regenerating rat liver suggesting that some newly synthesized chromosomal proteins preferentially associate with newly synthesized D N A as predicted by the segregation model. While the proteins were re
Archives of Oral Biology, 1982
Journal of Investigative Dermatology, 1996
Journal of Investigative Dermatology, 1994
Recently it has been shown that apolipoprotein E (apoE) secreted by keratinocytes in transplanted... more Recently it has been shown that apolipoprotein E (apoE) secreted by keratinocytes in transplanted epidermal grafts reaches the systemic circulation. In this study we ask which cells in cultures of epidermal keratinocytes, basal or suprabasal, are the source of apoE. By fractionating disaggregated cultures in gradients of Fico1l400, the small nondifferentiated cell s derived from the basal compartment were shown to be the source of apoE. The larger more differentiated cells derived from supra basal layers could not be shown to contain or secrete apoE, although they did contain the apoE mRNA.
Human Gene Therapy, 1994
Grafts of autologous keratinocytes genetically altered to secrete a new gene product are a potent... more Grafts of autologous keratinocytes genetically altered to secrete a new gene product are a potential vehicle for gene therapy. To consider the feasibility of such an approach, we have examined the ability of keratinocytes to secrete and deliver apolipoprotein E (apoE) to the circulation of mice bearing grafts of human keratinocytes. The grafted keratinocytes secreted two forms of apoE, an endogenous apoE encoded in the genome and a recombinant apoE encoded in a transfected gene construct. In vitro studies showed that endogenous apoE was secreted from basal keratinocytes whereas recombinant apoE was secreted from basal as well as suprabasal cells. On the basis of amounts of recombinant apoE present in the serum of grafted mice, we estimate that a graft occupying 2% of the surface area of an adult human would deliver 6.5-8.3 mg of recombinant apoE protein per day.
Proceedings of the National Academy of Sciences, 1998
Epidermis is renewed by a population of stem cells that have been defined in vivo by slow turnove... more Epidermis is renewed by a population of stem cells that have been defined in vivo by slow turnover, label retention, position in the epidermis, and enrichment in β 1 integrin, and in vitro by clonogenic growth, prolonged serial passage, and rapid adherence to extracellular matrix. The goal of this study is to determine whether clonogenic cells with long-term growth potential in vitro persist in vivo and give rise to a fully differentiated epidermis. Human keratinocytes were genetically labeled in culture by transduction with a retrovirus encoding the lacZ gene and grafted to athymic mice. Analysis of the cultures before grafting showed that 21.1–27.8% of clonogenic cells with the capacity for >30 generations were successfully transduced. In vivo , β-galactosidase (β-gal) positive cells participated in the formation of a fully differentiated epithelium and were detected throughout the 40-week postgraft period, initially as loosely scattered clusters and later as distinct vertical ...
DNA Damage and Repair in Human Tissues, 1990
There is considerable excitement about the possibility of somatic cell gene therapy, that is, the... more There is considerable excitement about the possibility of somatic cell gene therapy, that is, the introduction and expression of defined genes into cells for the purpose of providing a needed gene product. Most current research on gene therapy has been focused on the use of marrow stem cells as a therapeutic vehicle (Belmont et al., 1986). Inherited hematological disorders, such as severe combined immunodeficiency caused by adenosine deaminase deficiency, are diseases that might be amenable to this form of therapy (Kellems et al., 1985). A possible approach would be through genetic transfer of a recombinant adenosine deaminase gene into autologous marrow stem cells, transplantation of the transformed cells into the patient, and establishment of a population of stem cells that give rise to immunologically competent lymphoid cells. Although many questions need to be answered before we know the efficacy of such therapy, the National Institute of Health has issued guidelines for its use, and clinical trials have already begun (Culliton, 1989).
Journal of Investigative Dermatology Symposium Proceedings, 2004
For long-term cutaneous gene therapy, the therapeutic gene must be targeted to stem cells and be ... more For long-term cutaneous gene therapy, the therapeutic gene must be targeted to stem cells and be stably transmitted to and expressed in descendant cells. Retroviral vectors are highly efficient in gene transfer to human keratinocyte stem cells in culture; however, they cannot transduce quiescent stem cells in vivo. As lentiviral vectors (LVV) transduce non-proliferating cells, their ability to target human epidermal stem cells was evaluated. LVV were highly efficient in gene transfer to clonogenic keratinocytes in vitro. Despite higher transgene DNA content and comparable levels of transgene mRNA, levels of transgene product directed by lentivectors were 3folds lower than that of retrovectors. When transduced keratinocytes were grafted onto mice, transgene expression persisted for at least 20 wk; however, transgene product was detected primarily in the uppermost layers of epidermis. Inclusion of an element that is known to facilitate nuclear export of intron-less transcripts, resulted in enhanced transgene expression in keratinocytes. In vivo transduction of xenografted human skin with these vectors resulted in efficient gene transfer to epidermal progenitor cells. These results demonstrate stem cell transduction by LVV and point out the utility of using these vectors for direct gene transfer to and sustained expression in human epidermis.