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M C Lin

Publications and source records attributed to M C Lin.

At least 199 records · Page 11Linked to original sources
PubMed1987

Induction of glucagon sensitivity in a transformed kidney cell line by prostaglandin E2 and its inhibition by epidermal growth factor.

A model system using a transformed dog kidney cell line (Madin-Darby canine kidney), has been established for studying the process of differentiation. Glucagon responsiveness can be restored to these transformed cells by various differentiation inducers, including prostaglandin E2. Glucagon response was measured in terms of the ability of glucagon to stimulate cAMP production. Induction of glucagon sensitivity seems to be mediated by cAMP. The ability of various prostaglandin analogs to elevate the cAMP level correlates closely with their ability to induce glucagon sensitivity. In fact, 8-Br-cAMP is also a potent inducer. To define the nature of this cAMP-mediated process, we identified several inhibitors of this induction process. These differentiation inhibitors include serum, phorbol ester, and epidermal growth factor. These inhibitors do not have a direct effect on cAMP production by cells in the presence or absence of hormones. Furthermore, induction by 8-Br-cAMP is also inhibited by these agents. Therefore, the site of inhibition is located beyond the point of cAMP production. Possible interaction between cAMP- and epidermal growth factor-dependent phosphorylations is discussed.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1987

Decreased potency of glucagon on transformed-induced MDCK cells does not reflect an alteration of adenylate cyclase components.

The selective loss of glucagon sensitivity of transformed MDCK cells can be restored by differentiation inducers, a process which requires RNA and protein synthesis and glycosylation. Although the glucagon dose-response curve of normal MDCK cells resembled that of liver and kidney (Kact = 10 nM), the transformed-induced cells were 10-fold less sensitive to the hormone [activation constant (Kact) = 100 nM]. Additionally, the stimulation of cAMP synthesis by a glucagon fragment (glucagon) in transformed-induced cells was greatly reduced compared to normal cells. The adenylate cyclase regulatory components of transformed-induced MDCK cell membranes seemed unaltered compared to the parental line. Both contained equivalent amounts of cholera and pertussis toxin substrates, and soluble extracts were equally capable of reconstituting isoproterenol responsiveness of S49 cyc- membranes. However, membrane fusion studies demonstrated that the glucagon sensitivity of transformed-induced membranes could not be reconstituted with heterologous membranes. When donor transformed-induced membranes (with inactivated adenylate cyclase) were fused with acceptor HeLa membranes (normally unresponsive to glucagon and prostaglandin E), such hybrids were unresponsive to glucagon, although responsiveness to prostaglandin E was evident. Parallel hybrids with normal MDCK membranes were responsive to both glucagon and prostaglandin E. This difference could not be explained by an inhibitory effect of transformed-induced membranes on receptor-adenylate cyclase coupling under the fusion conditions: the ability of these membranes to serve as an acceptor for the reconstitution of vasoactive intestinal peptide responsiveness was identical to that of normal MDCK cells. The data suggest that the glucagon sensitivity induced in transformed MDCK cells differs significantly from that of the parental line. However, these differences cannot be explained by alterations of transformed-induced membrane components relevant to the coupling of hormone receptors to adenylate cyclase.

Adenylyl Cyclases↗
PubMed1986

Revertants of Ha-MuSV-transformed MDCK cells express reduced levels of p21 and possess a more normal phenotype.

Four subclones of the originally cloned Harvey murine sarcoma virus-transformed Madin Darby canine kidney (MDCK) cells have been isolated. These subclones fall into two general classes. Two subclones have a fibroblastic morphology, have lost the growth requirement for prostaglandin E1 (PGE1), do not respond to glucagon or vasopressin, and, in general, appear transformed. Two other subclones have epithelioid morphologies, are growth-stimulated by PGE1, respond to vasopressin with an increase in intracellular cAMP. We propose that these cells represent revertants to a more non-transformed phenotype. Unlike normal cells, however, these revertants grow under anchorage-independent conditions, express detectable but reduced amounts of the transforming gene product, p21, and grow in nude mice. The appearance of such revertants may be one cause of the observed heterogeneity of tumor cells.

Animals↗
PubMed1986

The effect of viral transformation on prostaglandin production depends on cell type.

The role of prostaglandins in cellular differentiation and transformation has been widely studied. We have found previously that prostaglandin E2 production was greatly diminished in dog kidney cells (MDCK) after transformation by Harvey murine sarcoma virus. In the present study, we have shown that viral transformation can have differing effects in the ability to modify the production of prostaglandin in cultured cells. For example, the prostaglandin E2 production in rat kidney cells (NRK) is decreased after transformation by Rous sarcoma virus, while production in 3T3 cells is increased markedly after transformation by the same virus. Similarly, SV40 transformation increases prostaglandin E2 production of 3T3 cells and decreases the production in rat thyroid cells (FRTL). These results indicate that the biosynthetic pathway for prostaglandin production has varying susceptibility following viral transformation and the effect of transformation depends more on the type of cell than virus. Taking advantage of the well-defined transforming proteins encoded by polyomavirus, we have further studied the relationship between prostaglandin production in cells and the expression of T antigens in transformed cells. We showed that the expression of middle T antigen, which is associated with a protein kinase and is responsible for phenotype of transformed cells, is required for the change in prostaglandin production in cells. How these changes of prostaglandin production relate to the progression of viral transformation remains to be explored.

Animals↗
PubMed1986

[Evaluation of a self-prepared enzyme immunoassay kit for detection of alpha-fetoprotein].

A "sandwich" technique of enzyme immunoassay (EIA) kit for human alpha-fetoprotein has been developed by using commercially available reagents. The assay range is 0.5-60 ng/ml. Within-run coefficients of variation (C.V.) for 20 determination at three different concentrations were 4.4-10.3%, and between-run C.V. were 4.9-10.8%. The sensitivity and specificity of the kit has been compared with those of two commercially available kits (Abbott and Roche r1 = 0.991, r2 = 0.989.) This kit has some advantages over currently available kits. The assay is incubated in room temperature, no waterbath is required. The solid phase is dry store, it have long shelf life. The kit is inexpensive to operate, and it is suitable for any laboratory.

Enzyme-Linked Immunosorbent Assay↗
PubMed1985

Glucagon-stimulated phosphorylation of rat liver glycogen synthase in isolated hepatocytes.

Addition of glucagon (20 nM) to the isolated hepatocytes from 24-h starved male rats results in an inactivation of glycogen synthase. The A0.5 for glucose-6-P is increased 2-fold over the control but the S0.5 for UDP-glucose is not significantly affected. The glucagon-stimulated inactivation of glycogen synthase is also accompanied by a 60-120% increase in the phosphorylation of the synthase. Glycogen synthase labeled with 32P by incubation of the hepatocytes with [32P] PO4(3-) was recovered by immunoprecipitation and the resulting immunoprecipitate was subjected to tryptic digestion. Analysis of the 32P-labeled peptides reveals that the sites corresponding to those phosphorylated by cAMP-dependent protein kinase and glycogen synthase (casein) kinase-1 (Itarte, E., and Huang, K.-P. (1979) J. Biol. Chem. 254, 4052-4057) are rapidly phosphorylated in response to glucagon. These results demonstrate that glucagon not only triggers the activation of cAMP-dependent protein kinase through an increase in the intracellular level of cAMP but also, by an unknown mechanism, activates a Ca2+- and cAMP-independent protein kinase.

Animals↗
PubMed1985

Expression of glucagon sensitivity by transformed MDCK cells normally unresponsive to glucagon: early commitment to differentiation.

A cloned line of canine kidney cells (MDCK) transformed with Harvey murine sarcoma virus, in contrast to the parental, untransformed line, expressed glucagon sensitivity only under controlled culture conditions. The glucagon sensitivity of transformed MDCK cells appeared after 10 days of culture if plated at less than 100,000 cells/dish or after 3 days if cells were plated at greater than 300,000 cells/dish. As there was no effect of conditioned medium from glucagon-sensitive cells on insensitive cells, media components seemed not to be involved in this phenomenon. Glucagon sensitivity appeared more readily in defined as opposed to serum-containing medium. In fact, as little as 2% fetal bovine serum inhibited the expression of glucagon sensitivity when included in defined medium over the course of the experiment. Furthermore, when transformed MDCK cells were exposed to serum for only the first 24 hr of culture, glucagon sensitivity on day 11 was identical to that of cells exposed to serum throughout the entire experiment. In contrast, exposure to serum later in culture (days 4-8) had no inhibitory effect on the expression of glucagon sensitivity on day 11. The data suggest that differentiation, or glucagon sensitivity, occurs when transformed, glucagon-insensitive cells achieve a critical high density and that differentiation is sensitive to inhibition by serum only during the first 24 hr of culture.

Animals↗
PubMed1982

Loss and restoration of glucagon receptors and responsiveness in a transformed kidney cell line.

A kidney cell line (MDCK) retains an adenylate cyclase system sensitive to glucagon, vasopressin, isoproterenol and prostaglandin E1. The stimulatory effect of glucagon on cAMP production was selectively lost in a cloned line derived from MDCK cells transformed by Harvey murine sarcoma virus. Sensitivity to glucagon was largely restored by treatment of the transformed cells with prostaglandin E1 or butyrate. Loss and reappearance of glucagon receptors seemed to be responsible for the observation. The parental MDCK line produced prostaglandins and in the transformed line, this function was abolished. These observations suggest that synthesis of glucagon receptors is controlled by endogenously produced prostaglandin in MDCK cells and that loss of glucagon receptors and their responsiveness in the transformed cells occurs as a consequence of the inability of these cells to synthesize this prostaglandin.

Animals↗
PubMed1980

Selective effects of organic mercurials on the GTP-regulatory proteins of adenylate cyclase systems.

Treatment of membranes from HeLa cells, rat adipocytes, and rat liver with organic mercurials results in complex effects on adenylate cyclase activity that are not mimicked by the reversible sulfhydryl reagent, tetrathionate. At low concentrations (0.1 mM or less 1 mercurials inactivate the enzyme; inactivation is reversed by the thiol-reducing agent, dithiothreitol. Treatment with higher concentrations of organic mercurials (1 mM and above) results in a time-dependent, irreversible change in the ability of guanine nucleotides and fluoride ion to stimulate adenylate cyclase activity. The irreversible changes are blocked by treatment of membranes with cholera toxin and NAD, suggesting that the GTP-regulatory component is the site of mercurial action. This is further suggested by the lack of irreversible effects of mercurials on adenylate cyclase activity in membranes from mouse lymphoma cells that lack this component. Irreversible effects of mercurials on the adipocyte cyclase system also include enhancement of basal activity and potentiation of the inhibitory effects of GTP on cyclase activity; the latter effects of GTP are mediated through a process independent from that mediating stimulation of activity by GTP. It is concluded that the GTP-regulatory proteins responsible for the modulation of adenylate cyclase activity by hormones and neurotransmitters contain the sites of action of organic mercurials. Their possible mode of action is discussed.

Adenylyl Cyclases↗
PubMed1980

Inhibition of cholera toxin activation of the adenylate cyclase system in intact HeLa cells.

Cholera toxin treatment activates the adenylate cyclase in intact HeLa cells. However, pretreatment of the cells with chemicals known to inhibit receptor internalization and lysosomal processing blocks the toxin activation. The agents found to inhibit the effect of cholera toxin include methylamine, ammonium chloride, chloroquine and dansylcadaverine. These chemicals did not affect either the binding of )125I)-cholera toxin to HeLa cells nor the ability of A1 peptide to activate the adenylate cyclase in plasma membrane preparations. We conclude that these chemicals act on the processing of the toxin subsequent to its binding and that internalization and lysosomal processing mediate the release of the active fragment from cholera toxin, which activates the adenylate cyclase system.

Adenylyl Cyclases↗