Biomedical subjects

R E Scott

Publications and source records attributed to R E Scott.

At least 55 records · Page 3Linked to original sources

PubMed1990

Proliferation and differentiation characteristics of normal human squamous mucosal cells of the upper aerodigestive tract.

Normal human squamous mucosal cells of the soft palate, buccal surface, epiglottis, hypopharynx, floor of the mouth, and tongue were cultured in vitro in serum-free medium. In medium MCDB 153 containing epidermal growth factor, insulin, bovine pituitary extract, and 0.1 or 2.0 mmol/L Ca++, squamous mucosal cells double every 24 hours. These cells then can be induced to arrest their proliferation reversibly by treatment with transforming growth factor-beta or ethionine, and they can irreversibly growth-arrest during senescence or when cultured in growth factor-deficient medium containing 2 mmol/L Ca++. The latter medium also induces differentiation, as does culture of cells in serum-containing medium. Serum-containing medium furthermore promotes extensive cell stratification and the formation of multilayered squamous mucosal tissue specimens that can be removed intact by Dispase treatment. These specimens represent potential autogenous mucosal grafts that can be used in patients who require reconstructive surgery of the oral cavity and oropharynx. Normal human squamous mucosal cells therefore closely resemble keratinocytes derived from the epidermis in the mechanisms that regulate proliferation and differentiation. This model cell system should facilitate future studies on upper aerodigestive tract squamous mucosal cell physiology and pathophysiology.

Calcium↗

PubMed1990

Developmental changes in levels of proopiomelanocortin intron A-containing heterogeneous nuclear RNA and mature messenger RNA in the anterior and neurointermediate lobes of the rat pituitary.

The POMC cells of the rat pituitary undergo dynamic phenotypic changes during differentiation. Here we have determined that alterations in the relative levels of a POMC precursor RNA species and POMC mRNA occur during development and may represent another level at which the POMC phenotype is developmentally regulated. We performed solution hybridization/nuclease protection assays using a POMC exon 1/intron A splice junction probe to quantitate levels of both intron A-containing POMC heterogeneous nuclear (hnRNA) and fully processed POMC mRNA in separated anterior and neurointermediate lobes of the fetal, neonatal, and adult pituitary. The levels of POMC hnRNA per anterior lobe increased 7-fold from embryonic day 15 to adulthood (0.022 to 0.159 fmol/lobe), while POMC mRNA levels increased 121-fold (0.15 to 18.2 fmol/lobe). POMC hnRNA levels per neurointermediate lobe increased 23-fold from embryonic day 18 to adulthood (0.024 to 0.54 fmol/lobe), while POMC mRNA levels increased 69-fold (0.65 to 44.6 fmol/lobe). Thus, both anterior and neurointermediate lobes contain higher relative abundances of POMC hnRNA compared to mRNA during early development. These subsequently decrease (from 1:7 to 1: approximately 110 in the anterior lobe and from 1:27 to 1:83 in the intermediate lobe over the ages examined) as the levels of POMC mRNA in both anterior and neurointermediate lobe increase at a greater rate than POMC hnRNA as development progresses. These results provide the first measurements of POMC mRNA and hnRNA levels during ontogeny and suggest that there may be a developmental change in the regulation of POMC primary transcript processing.

Animals↗

PubMed1990

Metaplastic change in mesenchymal stem cells induced by activated ras oncogene.

3T3 T murine mesenchymal stem cells have the potential to differentiate into a variety of different cell types even though they show a predilection to undergo adipocyte differentiation in vitro. The possibility that the activated c-Ha-ras (EJras) oncogene might influence the pathway of differentiation of these stem cells is investigated in the current study. Activated ras oncogene was transfected and stably expressed in 3T3 T cells; assays then were performed to determine its effect on differentiation. The results show that all EJras-transfected cell lines lose their ability to differentiate to adipocytes and instead differentiate into cells that express many characteristics of macrophages. Such cells contain numerous cytoplasmic granules, extensive nonspecific esterase activity, and anchorage-independent growth. The modulation of differentiation pathway from an adipocyte lineage to a macrophagelike cell lineage does not result from the transforming effect of EJras, because a nontransformed cell clone that expresses p21EJras protein also exhibits this modified differentiation pathway. These data suggest that the EJras oncogene specifically modulates the differentiation pathway of 3T3 T mesenchymal stem cells. This experimental system should therefore provide an excellent model to evaluate the mechanistic role of EJras in the process of metaplasia.

Adipose Tissue↗

PubMed1990

Stability of anticancer activity induced by cellular differentiation.

Human and murine cells can express anticancer activity which we define as any biologic process that can prevent or inhibit the expression of the transformed phenotype. The existence of anticancer activities has been demonstrated by many systems, including the fusion of normal cells with tumorigenic cells and the implantation of cancer cells into specific embryonic sites. Furthermore, cellular differentiation has recently been shown to regulate the expression of anticancer activity without limiting a cell's proliferative potential. Those results show that reversible nonterminal differentiation (NTD) induces 3T3T mesenchymal stem cells to become resistant to transformation by physical or chemical carcinogens or oncogene products and NTD induces spontaneously transformed 3T3T cells to revert to a benign state and to become resistant to retransformation. In addition, NTD induces SV40 T-antigen transformed 3T3T cells to revert to a nontransformed state that prevents growth in soft agarose. Data are now presented that define the relative stability of these differentiation-induced anticancer activities. Anticancer activity induced by NTD in 3T3T cells is shown to have a mean stability of 55 population doublings (PD) and a maximum stability of 88 PD. Anticancer activity induced by NTD in spontaneously transformed 3T3T cells shows a similar stability with a mean of 55 PD and a maximum of 95 PD. Finally, in SV40-3T3T cells, the induction of NTD suppresses soft agarose growth for a maximum of 20-30 PD. These results demonstrate that three forms of differentiation-induced anticancer activity are stable for an extended number of population doublings.

Animals↗

PubMed1989

Resistance to neoplastic transformation induced by nonterminal differentiation.

Cell clones derived from undifferentiated 3T3 T mesenchymal stem cells show a low rate of spontaneous transformation but can be efficiently transformed by a variety of carcinogens. In contrast, it is now reported that cell clones derived from nonterminally differentiated 3T3 T stem cells are highly resistant to neoplastic transformation induced by physical and chemical carcinogens. Differentiation-induced resistance to neoplastic transformation is evident in both in vitro and in vivo transformation assays and can be stably expressed for greater than 50 population doublings. These results establish that resistance to neoplastic transformation can be regulated in mammalian stem cells by the process of nonterminal differentiation.

Animals↗

PubMed1989

Nonterminally differentiated cells express decreased growth factor responsiveness.

In 3T3 T mesenchymal stem cells, at least four types of biological states exist that can mediate the control of cell differentiation and/or proliferation. These include the predifferentiation growth arrest state, the nonterminal differentiation state, the terminal differentiation state, and a growth arrest state induced by growth factor/serum deficiency. The current studies were performed to investigate the relative mitogenic responsiveness of cells at these four states and specifically to determine if nonterminally differentiated cells show decreased responsiveness to specific mitogens. Twenty-five different serum, plasma, and growth factor combinations were evaluated. The results show that undifferentiated, growth-arrested cells are highly responsive to numerous mitogens and that by definition terminally differentiated cells are not responsive to any mitogens. In contrast, nonterminally differentiated cells demonstrate a unique pattern of mitogenic responsiveness. Whereas nonterminally differentiated cells can be stimulated to proliferate by high concentrations of serum or plasma supplemented with growth factors, they cannot be stimulated to proliferate by combinations of multiple purified growth factors. These results suggest that the process of nonterminal differentiation is associated with a significant change in factors/cofactors required to stimulate cell proliferation and that these factors/cofactors are present in plasma.

Animals↗

PubMed1989

Role of the clinical laboratory in the diagnosis and management of malignant melanoma.

The biosynthesis of melanin from tyrosine is reviewed as the basis for assessment of laboratory tests that might potentially aid in the diagnosis and management of patients with malignant melanoma. These tests include qualitative and quantitative assays for the intermediates in metabolism of melanin and catecholamines, enzyme assays, metal ion analyses, and, most recently, immunoassays. Although currently no role exists for the clinical laboratory in the early diagnosis of malignant melanoma, serial quantitative analyses of total or individual melanogens or of catecholamine metabolites in urine or plasma specimens may be of value in the management of patients with this disorder. Immunologically based methods for the diagnosis and management of malignant melanoma hold some promise for the future.

Antigen-Antibody Complex↗

PubMed1989

Nonterminal differentiation represses the neoplastic phenotype in spontaneously and simian virus 40-transformed cells.

The potential of nonterminal cellular differentiation to stably repress the expression of the neoplastic phenotype of transformed cells is established. Nonterminal differentiation induces spontaneously transformed 3T3 T cells to revert to a nontransformed state and induces the revertant cell clones to become resistant to retransformation by UV irradiation or 4-nitroquinoline oxide treatment. Nonterminal differentiation also induces simian virus 40-transformed 3T3 T cells to repress expression of the large tumor antigen and to revert to a nontransformed state. Although the molecular mechanisms that mediate these and other forms of anticancer activity have not been definitively established, data are presented which suggest that differentiation-induced repression of large tumor antigen expression can be regulated at the level of transcription and/or RNA processing.

Animals↗

PubMed1989

Loss of proliferative potential during terminal differentiation coincides with the decreased abundance of a subset of heterogeneous ribonuclear proteins.

The decrease in abundance of a subset of highly conserved basic nuclear proteins is established to correlate with the loss of proliferative potential in association with the process of terminal differentiation in murine mesenchymal stem cells and human keratinocytes. These proteins, designated P2Ps for proliferation potential proteins, have apparent molecular masses of 30-40 kD, are associated with the 30-40S substructures of nuclear hnRNP complexes, and are recognized by antibodies made against core proteins of hnRNP particles. They also share an epitope in common with heat shock protein-90 (hsp90) and are recognized by two mAbs against hsp90. Two-dimensional electrophoretic Western blots furthermore show that P2Ps make up a subset of hnRNP proteins. Cells that possess these proteins express the potential to proliferate whether or not they are traversing the cell cycle. These include rapidly growing cells, reversibly growth-arrested cells, and nonterminally differentiated cells. In contrast, cells that have irreversibly lost their proliferative potential, such as terminally differentiated cells, show a marked reduction in the abundance of P2Ps as determined by immunodetection on Western blots. A correlation, therefore, exists between the presence of this subset of nuclear proteins and the proliferative potential in two cell types. These results raise the possibility that as a subset of hnRNP proteins, P2Ps may mediate posttranscriptional control of the processing of specific RNAs required for cell proliferation.

Animals↗

PubMed1989

Inhibition of simian virus 40 T-antigen expression by cellular differentiation.

Murine 3T3T stem cells transfected with pSV3neo DNA were employed to study the effects of somatic cell differentiation on simian virus 40 (SV40) T-antigen expression. This experimental approach was used because the 3T3T cell line is a well-characterized in vitro adipocyte differentiation system and the pSV3neo plasmid contains the early region of the SV40 genome and a selective marker, G418 resistance. Cell clones containing stably integrated pSV3neo which expressed T antigen were isolated in G418-containing medium. Most of these cell clones differentiated poorly. However, several clones retained the ability to efficiently differentiate into adipocytes, and with these cell clones, it was established that adipocyte differentiation markedly repressed T-antigen expression. The differentiation-specific repression of T-antigen expression did not result from a loss of proliferative potential associated with terminal differentiation, because it was observed in adipocytes that could be restimulated to proliferate. In such cells, restimulation of cell growth induced reactivation of T-antigen expression. Repression of T-antigen expression was also demonstrated during differentiation of SV40 T-antigen-immortalized human keratinocytes. These results establish that the process of cellular differentiation can repress T-antigen expression in at least two distinct biological systems.

Adipose Tissue↗

PubMed1989

Integrated control of proliferation and differentiation of mesenchymal stem cells.

The physiological control of cellular proliferation and differentiation is an integrated regulatory process. This conclusion is based upon observations using numerous in vivo and in vitro experimental systems of which murine BALB/c 3T3 T mesenchymal stem cells represent an excellent in vitro model. In these cells the coupling of growth arrest and differentiation occurs at a distinct biological state, and this predifferentiation arrest state is distinguishable by a variety of criteria from other restriction points, such as the growth factor deficiency arrest state and the nutrient deficiency arrest state. Most importantly, only cells at this predifferentiation arrest state acquire the potential to differentiate without undergoing DNA synthesis. From this state, differentiation can then occur as a two-step process. Cells first undergo nonterminal differentiation and, second, they terminally differentiate. Nonterminal differentiation is characterized by expression of a completely differentiated adipocyte phenotype with retention of proliferative potential. Thereafter, when nonterminally differentiated cells undergo the terminal event in differentiation, they irreversibly lose their proliferative potential. In this paper, data are reviewed which establish that the integrated control of proliferation and differentiation in 3T3 T mesenchymal stem cells is mediated both at the predifferentiation arrest state and at the state of nonterminal differentiation.

Animals↗

PubMed1988

Ability of normal human keratinocytes that grow in culture in serum-free medium to be derived from suprabasal cells.

The current studies were performed to determine from which regions of the skin keratinocytes that grow in vitro are derived. Normal human foreskin specimens were first separated by differential trypsinization into two suprabasal fractions and one basal fraction. Utilizing complete MCDB 153 basal nutrient culture medium containing epidermal growth factor and insulin, we then evaluated the clonogenic potential of cells in these three fractions. Suprabasal cell fractions demonstrated a colony-forming efficiency as great as or greater than that of the basal cell fraction, and 10%-15% of the keratinocytes that grew in primary and secondary cultures expressed involucrin, a suprabasal keratinocyte differentiation marker. Of such involucrin-containing keratinocytes, 80% also possessed the potential to undergo DNA synthesis, as determined by autoradiography following a 48-hour incubation with [3H]thymidine. These observations support the conclusion that the human keratinocytes that grow in vitro in serum-free medium can be derived from suprabasal cells and, therefore, that a state of nonterminal keratinocyte differentiation exists.

Cell Division↗

PubMed1988

Tumor necrosis factor inhibits the terminal event in mesenchymal stem cell differentiation.

Control of the terminal event in cellular differentiation is an important normal regulatory process, and the expression of defects in the control of this process has been implicated in the pathogenesis of cancer. To determine if tumor necrosis factor (TNF), which is an important biological response modifier, can inhibit terminal differentiation, we have studied 3T3 T mesenchymal stem cells. This experimental cell system was employed because a well-defined series of steps in differentiation has been defined and cells at each stage of differentiation can be isolated. For example, nonterminal differentiated cells can be isolated, and their transition to a terminal differentiation state can be evaluated. The most interesting results in the current studies show that TNF blocks the terminal event in mesenchymal stem cell differentiation. Inhibition of the terminal event of differentiation by TNF is reversible and is not associated with inhibition of selective or general protein synthesis. Evidence is also presented that cell clones that are defective in their ability to undergo the terminal event in differentiation secrete factor(s) that inhibit the terminal event in differentiation. These observations suggest that the inhibition of the terminal event in differentiation may be mediated via autocrine or paracrine regulatory molecules such as tumor necrosis factor.

Adipose Tissue↗

PubMed1988

Differentiation, cancer, and anticancer activity.

Carcinogenesis is a multistep process that results from the development of a variety of defects in the control of differentiation and proliferation. To investigate this concept further, 3T3 T mesenchymal stems cells were employed to establish that a distinct sequence of biological processes is involved in the control of differentiation and proliferation, and that these processes are integrally regulated. Specific defects in these regulatory processes were next established as being involved in carcinogenesis. These defects, however, were found not to be absolute; rather, they appear to involve changes in the stringency by which differentiation and proliferation are integrally regulated. Finally, it was established that when normal or transformed stem cells are induced to undergo nonterminal differentiation (which is one step in the integrated control of proliferation and differentiation), they can be made resistant to carcinogenesis or to revert to a nontransformed state. These data provide strong evidence that a critically important requirement for normal homeostasis is maintenance of intact cellular mechanisms to integrally regulate differentiation and proliferation.

Animals↗

PubMed1988

Biologic mechanisms for the regulation of normal human keratinocyte proliferation and differentiation.

Normal human keratinocytes can be grown in serum-free medium, and the integrated control of their proliferation and differentiation can be modulated experimentally. The growth of cultured human keratinocytes can also be specifically arrested at either reversible or irreversible growth arrest states. Reversible growth arrest is induced by culture in medium containing TGF-beta or ethionine or in medium deficient of isoleucine. Irreversible growth arrest is induced by culture in razoxane-containing medium or by routine passage of keratinocytes until senescence results. The current studies were performed to determine from which growth arrest states keratinocyte differentiation occurs. Cells were therefore growth-arrested at each state, and they were then incubated in several different differentiation-promoting culture conditions. The results show that differentiation, as determined by morphologic, cytochemical, and immunofluorescent assays, can be induced from multiple reversible and irreversible growth arrest states by a series of complex biologic mechanisms. More specifically, at least three distinct stages appear to be involved in the process of keratinocyte differentiation. First, cells arrest their growth at a reversible predifferentiation state. Second, cells irreversibly lose their proliferative potential. Finally, cells express the terminally differentiated keratinocyte phenotype.

Antibodies, Monoclonal↗

PubMed1988

Two subtypes of reversible cell cycle restriction points exist in cultured normal human keratinocyte progenitor cells.

Three methods can induce reversible arrest of the growth of cultured human keratinocytes in the G1 phase of the cell cycle. These include the incubation of cells in medium containing transforming growth factor (TGF)-beta or ethionine, or in isoleucine-deficient medium. The current studies were performed to determine if the growth arrest induced by these methods occurs at a common or at a distinct G1 state(s). We first evaluated the relative time interval required for arrested cells to initiate DNA synthesis after growth restimulation with mitogenic medium. The results show that ethionine arrested cells require 22 to 28 hours to initiate DNA synthesis and that a maximum rate of DNA synthesis occurs at 46 hours. Cells arrested by isoleucine deficiency required 10 to 12 hours to initiate DNA synthesis with peak DNA synthesis occurring at 24 hours. Finally, TGF-beta arrested cells require only 6 to 8 hours to initiate DNA synthesis and show a maximum rate of DNA synthesis at 18 hours. We next evaluated if cells that were growth arrested at these states were differentially capable of initiating DNA synthesis in different types of potentially mitogenic medium. The results show that if TGF-beta arrested cells were refed TGB-beta free serum containing medium with ethionine or similar medium with isoleucine deficiency, no DNA synthesis occurred. In contrast, if cells whose growth was arrested in ethionine-containing medium or in isoleucine-deficient medium were refed mitogenic medium containing TGF-beta, significant DNA synthesis was detected. These results suggest that least two different types of reversible growth arrest states exist in cultured human keratinocytes. One appears to be mediated by receptor-dependent processes, such as that induced by TGF-beta, and the other appears to be mediated by other types of metabolic events, such as those induced by ethionine treatment or by isoleucine deficiency.

Cell Cycle↗

PubMed1988

Human squamous carcinoma cells express complex defects in the control of proliferation and differentiation.

Four human squamous carcinoma cell (SCC) lines (SCC-9, SCC-13, SCC-15, and SCC-25) were studied to characterize their relative ability to control proliferation and differentiation. These experiments were based on previous data that established that in normal human keratinocytes three distinct and sequential steps are involved in the integrated control of proliferation and differentiation: 1) reversible growth-arrest at a predifferentiation state, 2) irreversible loss of proliferative potential, and 3) terminal differentiation. The current results show that SCC can show changes in the culture conditions required to undergo reversible growth-arrest and SCC can express partial or complete defects in their ability to irreversibly growth-arrest or terminally differentiate. For example, SCC-9 and SCC-25 cannot irreversibly growth-arrest or terminally differentiate, SCC-13 can irreversibly growth-arrest but cannot terminally differentiate, and SCC-15 can irreversibly growth-arrest and terminally differentiate to a moderate extent. These results therefore extend previous data by establishing that the malignant transformation of human epithelial cells does not simply result from defects in the control of terminal differentiation but rather from a combination of complex defects in the regulation of proliferation and differentiation.

Calcium↗

PubMed1988

Aproliferin: a modulator of proliferative potential.

Cellular differentiation proceeds through a series of steps in which cells undergo modifications in cellular phenotype and proliferative potential. Differentiation has been extensively studied in 3T3-T mesenchymal stem cells and growth arrest, non-terminal and terminal differentiation have been identified as three distinct stages in the adipocyte differentiation of these cells. The terminal stage of differentiation is associated with irreversible loss of proliferative potential and commitment to the expression of the adipocyte phenotype. A protein has been partially purified from human plasma that can induce the transition of 3T3-T adipocytes from the non-terminal to the terminal state of differentiation. This protein, designated aproliferin, has a mol. wt of approximately 45,000 and is trypsin, acid and heat labile. Induction of terminal differentiation by aproliferin is associated with changes in the synthesis of a limited number of cellular proteins. The ability of aproliferin to induce terminal differentiation in non-terminally differentiated cells is highly specific as a wide variety of pharmacological and biochemical agents do not mimic the effects of this agent. Apoliferin may be one of an emerging class of molecules which can affect differentiation and induce irreversible changes in cell function.

Animals↗