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Biomedical subjects

M I Sherman

Publications and source records attributed to M I Sherman.

At least 37 records · Page 2Linked to original sources
PubMed1987

Differential uptake, binding, and metabolism of retinol and retinoic acid by 10T1/2 cells.

Previous studies have shown that all-trans-retinoic acid fails to inhibit chemically induced transformation in 10T1/2 cells except at toxic levels, whereas retinol and many synthetic retinoids are potent inhibitors. In contrast, in many systems retinoic acid is a more effective modulator of differentiation and carcinogenesis than is retinol. In any attempt to explain this anomaly, we have studied the differential metabolism of retinoic acid and retinol by 10T1/2 cells and by their initiated and transformed derivatives, and have also reexamined these cells for the presence of retinoid-binding proteins. Whereas retinoic acid was depleted from the medium bathing 10T1/2 and initiated 10T1/2 cells within 48 h of treatment, retinol was concentrated 500-fold by these cells, and disappeared from the culture medium no faster than from cell-free medium. Retinoic acid metabolism by a number of transformed cell lines varied widely. There was no apparent correlation between metabolizing ability and transforming agent (methylcholanthrene, X-rays, fission spectrum neutrons, and plasmid oncogene transfection). Uptake of retinoic acid was seen in all cell lines and was not correlated with its metabolism. Retinol was metabolized minimally by all cell lines tested; metabolism of retinol was not correlated with retinoic acid metabolizing ability. Retinoic acid-induced growth inhibition and cytotoxicity were not correlated with metabolizing ability, suggesting that the rate of metabolism of retinoic acid is not a major determinant of its acute biological effects. Using sensitive radioimmunoassays, cellular retinoic acid- (CRABP) and retino-binding proteins (CRBP) were both detected in 10T1/2 and initiated 10T1/2 cells. CRABP levels of about 16 ng/10(6) cells were about 4-fold higher than CRBP levels. Therefore, lack of CRABP does not explain the failure of retinoic acid to inhibit carcinogen-induced transformation in these cells. These studies suggest that the inability of retinoic acid to inhibit transformation in the 10T1/2 cell system may be due to its rapid metabolism and clearance from the medium. On the other hand, the high cellular uptake and stability of retinol in these cells could be an important factor in the inhibition of neoplastic transformation by this retinoid.

Carrier Proteins↗
PubMed1987

Analyses of the interactions between retinoid-binding proteins and embryonal carcinoma cells.

[3H]Retinoic acid (RA) and [3H]retinol bind in an unsaturable manner to isolated nuclei from Nulli-SCC1 and PCC4.aza1R embryonal carcinoma (EC) cells. When nuclei are challenged with the same labeled retinoids on their respective binding proteins (CRABP and CRBP), much less binding is observed and the binding is saturable. RA-CRABP does not compete with [3H]retinol-CRBP for binding to specific Nulli-SCC1 nuclear sites, whereas retinol-CRBP (but not apo-CRBP) actually potentiates the binding of [3H]RA-CRABP to these nuclei. The binding of [3H]RA-CRABP and [3H]retinol-CRBP is not dramatically affected by prior removal of the outer nuclear membrane with Triton X-100. However, treatment with the detergent after the binding reaction is complete removes about half of the bound [3H]RA-CRABP and almost all of the bound [3H]retinol-CRBP. We measured specific retinoid-binding activities in nucleoplasmic extracts of Nulli-SCC1 and PCC4.aza1R cells. The only readily detectable specific binding activity in nucleoplasmic extracts from untreated cells was for [3H]retinol in PCC4.aza1R preparations. Nucleoplasmic extracts from Nulli-SCC1 and PCC4.aza1R cells pretreated with RA had considerable levels of specific [3H]RA-binding activity with little or no increase in [3H]retinol binding. By contrast, similar extracts from Nulli-SCC1 cells treated with retinol bound large amounts of both [3H]retinol and [3H]RA. Under the same conditions, PCC4.aza1R extracts also contained [3H]RA-binding activity with no increase in [3H]retinol binding above the high endogenous levels. Although these results might reflect translocation of binding proteins from cytoplasm to nucleus, other interpretations must be considered since we often observed an increase, rather than the expected reduction, in cytoplasmic retinoid-binding protein levels.

Animals↗
PubMed1986

Retinoic acid effect on cyclic AMP-dependent protein kinases in embryonal carcinoma cells: studies with differentiation-defective sublines.

Retinoic acid induces the differentiation of PCC4.aza 1R and Nulli-SCC1 embryonal carcinoma (EC) cells. In response to retinoic acid treatment, the levels of cyclic AMP (cAMP)-dependent protein kinases are enhanced in the plasma membrane within 17 hours and in the cytosol fractions of these cells within 2 to 3 days, as determined by phosphotransferase activity and by 8-azido-cyclic [32P]AMP binding to the RI and RII regulatory subunits. PCC4 (RA)-1 and Nulli (RA)-1 are mutant EC lines that fail to differentiate in response to retinoic acid. The former line, but not the latter, lacks cellular retinoic acid-binding protein (cRABP). Basal levels of cAMP-dependent protein kinase activities are elevated in PCC4 (RA)-1 cells. When these cells are treated with retinoic acid, neither cAMP-dependent protein kinase activities nor cAMP binding activities are enhanced; rather, there is a decrease in cytosolic kinase activity and RI subunit. On the other hand, Nulli (RA)-1 cells exhibit increases both in cAMP-dependent protein kinase activities and cAMP binding in response to retinoic acid. These results raise the possibility that cRABP mediates the enhancement of regulatory and catalytic subunits of cAMP-dependent protein kinases in both the membrane and the cytosolic fractions of the teratocarcinoma cells. There also might be some effects of retinoic acid on the cAMP-dependent protein kinase that are unrelated to differentiation and to the presence of cRABP.

Animals↗
PubMed1986

Reversible and irreversible effects of retinol upon the phenotypic properties of embryonal carcinoma cells.

We have generated an embryonal carcinoma cell line, NR1, which is not growth-inhibited in response to retinol. Although the retinol-treated cells undergo morphological change, show reduced levels of the surface antigen SSEA-1 and possess increased surface reactivity with antifibronectin serum, the extent of phenotypic change of NR1 cells in response to retinol is not so great as that following treatment with retinoic acid. Furthermore, unlike cells from lines such as F9, the retinol-promoted morphological alterations appear to be reversible. The increased adherence of NR1 cells to glass coverslips in the presence of retinol suggests that stronger interaction with the substratum is responsible for the observed alteration in appearance of the cells. It is possible that NR1 cells exposed to retinol progress no further than a reversible, early stage of differentiation. Alternatively, retinol-treated cells might express a group of markers normally associated with, or accompanying, an irreversible, differentiated phenotype even though the cells are not, in fact, undergoing differentiation.

Animals↗
PubMed1985

Metabolism of retinoids by embryonal carcinoma cells.

Several embryonal carcinoma (EC) cell lines were tested in culture for their ability to metabolize all-trans-[3H]retinol, all-trans-[3H]retinyl acetate, and all-trans-[3H]retinoic acid. There was little, if any, metabolism of all-trans-retinol to more polar compounds; we failed to detect conversion to acidic retinoids by reverse-phase high performance liquid chromatography and derivatization. We also did not observe [3H]retinoic acid when EC cells were incubated with [3H]retinyl acetate. Unlike the other retinoids, all-trans-[3H]retinoic acid, even at micromolar levels, was almost totally modified by cells from several EC lines within 24 h. Most of the labeled products were secreted into the medium. Some EC lines metabolized retinoic acid constitutively, whereas others had an inducible enzyme system. A differentiation-defective line, which contains little or no cellular retinoic acid-binding protein activity, metabolized retinoic acid poorly, even after exposure to inducers. At least eight retinoic acid metabolites were generated; many contain hydroxyl residues. Our data lead us to propose that retinol does not induce differentiation of EC cells in vitro via conversion to retinoic acid. Also, the relatively rapid metabolism of retinoic acid by EC cells suggests either that the induction of differentiation need involve only a transient exposure to this retinoid or that one or more of the retinoic acid metabolites can also promote differentiation.

Animals↗
PubMed1985

Role of retinoids in differentiation and growth of embryonal carcinoma cells.

To study how retinoids promote differentiation and inhibit proliferation of embryonal carcinoma (EC) cells, we have followed their intracellular fate. Retinoic acid (RA) is effectively metabolized to more polar compounds by many EC lines. Unlike RA, retinol is slowly metabolized. Our inability to detect conversion of retinol to RA might indicate that the two retinoids elicit their effects on EC cells in different ways. Retinol added to cultures quickly appears in the nuclear fraction; the proportion associated with nuclei after detergent extraction is initially very low but increases with time. Retinol and RA might be translocated to nuclei by their respective binding proteins [cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding protein (CRABP)]: isolated EC nuclei have specific, independent binding sites for both holoproteins but not their ligands. CRABP cannot be detected in the nucleoplasm of untreated EC cells, but activity is measurable after cells are exposed to RA. Interestingly, incubation with retinol promotes movement of both CRBP and CRABP into the nucleoplasmic fraction. Finally, we have demonstrated that brief exposure to RA dramatically reduces the cloning efficiency of EC cells. Since some cells are unaffected even by lengthy exposures to RA whereas the growth of their progeny is inhibited, we suggest that EC cells can become epigenetically refractory to RA.

Animals↗
PubMed1985

Isolation of embryonal carcinoma cell lines that express integrated recombinant genes flanked by the Moloney murine leukemia virus long terminal repeat.

We have used a producer NIH 3T3 cell line that secretes, together with the helper Moloney murine leukemia virus (Mo-MuLV), a transducing recombinant virus containing the neomycin-resistance gene linked to the Mo-MuLV long terminal repeat (LTR). By infecting three embryonal carcinoma cell lines, PCC4.aza1R, F9tk-, and Nulli-SCC1, with this recombinant virus, we have isolated many transductant clones that stably express the integrated neomycin-resistance gene. These clonal transductant lines consist of undifferentiated embryonal carcinoma cells as judged by morphology, tumorigenicity in 129/Sv mice, and cell-surface antigenic markers. Analysis of the integrated recombinant viral genes by Southern blot hybridization revealed that some of the lines have single copies, whereas others have multiple copies, probably in multiple sites. Although these transductant lines contained many copies of helper Mo-MuLV integrated in the cellular genome, expression of these helper viruses was not detected either by reverse transcriptase activity or by X-C plaque assay. Two F9tk--derived, G418-resistant transductant lines were superinfected with a second recombinant transducing virus that contains the herpes simplex virus thymidine kinase gene flanked by the Mo-MuLV LTR. The frequency of transduction to yield clones able to grow in hypoxanthine/aminopterin/thymidine medium was similar to that of the parental F9tk- cells. These results suggest that the expression of the neomycin-resistance gene, linked to MoMuLV LTR in the transductant embryonal carcinoma cell clones, is due to a cisacting mechanism(s).

Animals↗
PubMed1984

Complementation analyses of differentiation-defective embryonal carcinoma cells.

We have generated cell hybrids by fusing embryonal carcinoma (EC) cells which fail to differentiate in response to retinoic acid (RA) and/or hexamethylenebisacetamide (HMBA). The first two classes of hybrids were between an RA- line (also unresponsive to HMBA) that lacks cellular RA binding protein (cRABP) activity and HMBA- lines which possess cRABP and differentiate in the presence of RA. All of the hybrid clones possessed cRABP and differentiated normally upon exposure to either RA or HMBA. When the aforementioned RA- mutant was fused with a second mutant which was refractory to RA and HMBA but possessed cRABP activity, the resultant hybrid clones were responsive to both RA and HMBA and had cRABP activity. These results suggest that all of these mutants were recessive and complementary. Tumors from these hybrid lines differentiated extensively, in some instances much more so than the mutant parental lines and even the wild-type lines from which the mutants were derived. Based upon these observations, we propose that various EC lines might differentiate poorly in tumor form for different reasons. Hybrids between two differentiation-defective, cRABP- lines appeared to be at least partially complemented for responsiveness to RA and HMBA. These hybrids contained low but detectable levels of cRABP. This is not a consequence of tetraploidy since fusions between cells from the same mutant line retained their differentiation-defective phenotype and possessed little or no cRABP activity. Unlike tumors from the other hybrids described above, tumors from these hybrid lines expressed a very restricted pattern of differentiated cell types. This might be because the mutant lines in the latter hybrids originally derived from the same wild-type EC line.

Acetamides↗
PubMed1984

Sodium butyrate induces histone hyperacetylation and differentiation of murine embryonal carcinoma cells.

Cells from embryonal carcinoma (EC) lines 6050AJ and PCC4.aza 1R differentiate in response to treatment with sodium butyrate as well as retinoic acid (RA) or hexamethylenebisacetamide (HMBA). Murine 6050AJ EC cells exposed to sodium butyrate possess hyperacetylated forms of histones H4 and altered forms of histones H2a and H2b, whereas histones from cells treated with other inducers appear to be unaffected. These results might indicate that the mechanism by which sodium butyrate promotes differentiation of EC cells is different from the ways in which RA and HMBA act. Differentiation-defective PCC4(RA)-1 EC cells fail to respond to RA, presumably because they possess minimal amounts of active binding protein for RA (cRABP). Sodium butyrate treatment of these cells results in only a modest level of differentiation. On the other hand, exposure to sodium butyrate plus RA leads to extensive differentiation. As is the case with 6050AJ cells, PCC4(RA)-1 cells treated with sodium butyrate also contain hyperacetylated histones. Furthermore, these cells now possess high levels of cRABP. The latter observations suggest that sodium butyrate has the ability to reactivate a silent cRABP gene in PCC4(RA)-1 cells and thereby lead to extensive differentiation via the retinoid pathway when RA is added.

Acetylation↗
PubMed1984

Changes in protein synthetic profiles during retinoic-acid induction of differentiation of murine embryonal carcinoma cells.

Analysis of two-dimensional gel electrophoretic patterns of proteins labeled with [35S]methionine was undertaken to investigate changes in gene expression in embryonal carcinoma cell line Nulli-SCC1 during the induction of differentiation in vitro by retinoic acid. The results indicate that changes in protein profiles occur long before overt morphological differentiation is observed. Studies utilizing the antimetabolite, alpha-amanitin, coupled with those involving the cell-free translation of poly(A)+ -RNA, suggest that gene expression is controlled post-transcriptionally as well as transcriptionally. In this respect, embryonal carcinoma cells appear to resemble early embryonic cells, which show evidence of multiple levels of control of gene expression during the initial stages of development and differentiation.

Amanitins↗
PubMed1983

Differentiation-defective mutants of mouse embryonal carcinoma cells: response to hexamethylenebisacetamide and retinoic acid.

We have generated by mutagenesis eight differentiation-defective sublines from three murine embryonal carcinoma (EC) cell lines. These mutants grossly resemble parental cells in the absence of inducers of differentiation. Based upon response to retinoic acid (RA) or hexamethylenebisacetamide (HMBA), the mutants can be grouped into three types: (a) RA-selected cells that lack cellular RA binding protein (cRABP) activity and fail to differentiate in response to RA or HMBA; (b) RA- or HMBA-selected cells that possess cRABP but differentiate poorly, if at all, in the presence of RA or HMBA; and (c) cells originally selected for lack of response to HMBA but which retain cRABP and the ability to differentiate in response to RA.

Acetamides↗
PubMed1983

Murine embryonal carcinoma cells differentiate in vitro in response to retinol.

In an initial effort to determine whether circulating retinol might promote differentiation of embryonal carcinoma (EC) cells in tumor form, we have assessed the ability of retinol to stimulate differentiation of cultured EC cells. We found that retinol induces several murine EC cell lines to differentiate in vitro. Differentiated derivatives were distinguishable from parental EC cells by morphology, cell surface antigenic properties and levels of secretion of plasminogen activator. Retinol effects could be seen at concentrations as low as 8.7 X 10(-8) M (0.025 microgram/ml). Only two of eight EC lines tested failed to differentiate in response to retinol: PCC4-aza1R, which dies at retinol concentrations above 3.5 X 10(-7) M; and PCC4(RA)-1, a mutant line derived from PCC4-aza1R cells, which also fails to differentiate in response to retinoic acid.

Animals↗
PubMed1983

Retinoid binding protein activities in murine embryonal carcinoma cells and their differentiated derivatives.

It has been proposed that cellular retinoic acid binding protein is essential for retinoid-induced differentiation of embryonal carcinoma line PCC4.aza1R. To assess the generality of this proposal, we have tested for the presence of cellular retinoic acid binding protein activities in several other embryonal carcinoma lines. Cytosolic extracts from all cells were found to possess binding proteins for retinoic acid and also for retinol, although levels varied widely among the different lines. There was no clear quantitative relationship between binding protein activities and the propensity of the cells for differentiation in tumor form or under various in vitro conditions. Our results suggest that other factors might modulate the response of embryonal carcinoma cells to retinoids and/or that alternate pathways for differentiation which do not involve retinoids and retinoid binding proteins exist in these cells. When embryonal carcinoma cells are stimulated to differentiate, the derivatives can possess higher, lower, or similar levels of retinoic acid binding protein activity. These levels appear to reflect the phenotype of the differentiated cells rather than the conversion from a tumorigenic to a nontumorigenic state.

Animals↗
PubMed1983

Effects of arotinoids upon murine embryonal carcinoma cells.

Five arotinoids have been compared with all-trans- and 13-cisretinoic acids for their ability to promote differentiation of cells from murine embryonal carcinoma line Nulli-SCC1. Ro-13-7410, which contains a terminal carboxylic acid residue, and Ro-14-9572, the sodium sulfinate derivative, are potent inducers of differentiation. The sodium sulfonate derivative, Ro-14-3899, is somewhat less active, whereas the ethyl sulfone (Ro-15-1570) and Ro-15-0778, an arotinoid lacking a terminal group, have little or no effect on embryonal carcinoma cell differentiation. Competition by the arotinoids with all-trans-retinoic acid for sites on the cellular retinoic acid-binding protein is qualitatively consistent with their capacity for promoting differentiation. This relationship and the response of differentiation-defective embryonal carcinoma cells to Ro-13-7410 support the view that arotinoids and retinoids promote differentiation of embryonal carcinoma cells via the same mechanism.

Animals↗
PubMed1982

Protein synthetic requirements for the outgrowth of trophoblast cells from mouse blastocysts.

Mouse blastocysts cultured under optimal conditions attach to the surface of the culture dish and subsequently give rise to outgrowths of trophoblast cells. The migration of trophoblast cells appears to be analogous to their behavior during the invasive phase of implantation in utero. In these studies, we have attempted to determine the time of synthesis and nature of the products required for trophoblast outgrowth. Fourth-day blastocysts were cultured in two different media: cNCTC, a nutrient medium supplemented with fetal calf serum, and PCMF, a simple preimplantation medium lacking amino acids and serum. Trophoblast outgrowth occurs in the former, but not in the latter, medium. Most blastocysts will give rise to outgrowths following as little as 12 hr of exposure to cNCTC at the appropriate time, even if they are subsequently placed in PCMF. Outgrowth fails to occur if blastocysts are treated with cycloheximide during the interval of exposure to cNCTC, whereas treatment with the antimetabolite after the period of culture in cNCTC does not block outgrowth. These observations are consistent with the view that protein(s) essential for trophoblast outgrowth are synthesized many hours prior to the actual event.

Amino Acids↗
PubMed1982

Effect of antimetabolites on programming of inner cells of the mouse blastocyst.

When early blastocysts are subjected to immunosurgery, the resulting inner cell masses (ICMs) regenerate trophoblast cells. In contrast, ICMs from later blastocysts produce endoderm cells. We have found that if embryos are treated with cycloheximide during the transition from the early to late blastocyst stages, subsequently isolated ICMs give rise to trophoblast-like giant cells. These cells do not, however, exhibit detectable levels of delta 5, 3 beta-hydroxysteroid dehydrogenase, an enzyme normally found in primary trophoblast cells. Neither Colcemid nor alpha-amanitin affects the conversion of the ICM program in the same way as cycloheximide, although alpha-amanitin added at later stages interferes with the formation of a cohesive endoderm layer around the isolated ICM. We propose that the reprogramming of ICM cells involves at least two events: one terminates the giant cell program and the other, occurring later in development, promotes endoderm formation.

3-Hydroxysteroid Dehydrogenases↗