Biomedical subjects

B Korc-Grodzicki

Publications and source records attributed to B Korc-Grodzicki.

9 recordsLinked to original sources

PubMed1996

Effects of estradiol on the expression and production of IGFBP-2 by R3230AC mammary tumor cells.

The R3230AC mammary adenocarcinoma of the Fischer rat possesses Type I and Type II IGF receptors. The mRNAs for IGFBP-2, -3, -4, -5 and -6 have been recently identified in this tumor in vivo and in vitro. Using western blotting techniques on tumor tissue homogenates or conditioned media, we demonstrated that IGFBP-2 and, to a lesser extent, IGFBP-3 were expressed, produced, and secreted by the R3230AC tumor cells. Moreover, immunohistochemical assessment of tumor sections with anti-IGFBP-2 demonstrated that signal for IGFBP-2 was localized in the neoplastic glandular epithelium and often in the lumina of the pseudoglandular structures characteristic of this neoplasm. Expression of IGFBP-2 is regulated by the estrogen status of the host. The significant increase occurring in tumors from ovariectomized hosts was completely reversed with hormone repletion. Both mRNA expression and production of IGFBP-2 in vitro were also regulatable by the presence of estradiol-17 beta, with both processes being inhibited by its addition to the cell culture medium. Thus, the response of IGFBP-2 to estrogen showed agreement both in vivo and in vitro, whereas progesterone had no significant effect on these parameters. In the R3230AC tumor, estrogen treatment in vivo decreases tumor growth. Therefore, a relationship could exist between the action of estradiol to inhibit production of IGFBP-2 and the ability of estrogens to regulate tumor growth.

Adenocarcinoma↗

PubMed1993

Evidence for an extra-cellular function for protein kinase A.

In addition to its intra-cellular functions, cAMP-dependent protein kinase (PKA) may well have an extra-cellular regulatory role in blood. This suggestion is based on the following experimental findings: (a) Physiological stimulation of blood platelets brings about a specific release of PKA, together with its co-substrates ATP and Mg++; (b) In human serum, an endogenous phosphorylation of one protein (p75, M(r) 75 kDa) occurs; this phosphorylation is enhanced by addition of cAMP and blocked by the Walsh-Krebs specific PKA inhibitor; (c) No endogenous phosphorylation of p75 occurs in human plasma devoid of platelets, but the selective labeling of p75 can be reproduced by adding to plasma the pure catalytic subunit of PKA; (d) p75 was shown to be vitronectin (V), a multifunctional protein implicated in processes associated with platelet activation, and thus a protein whose function may require modulation for control; (e) The phosphorylation of vitronectin occurs at one site (Ser378) which, at physiological pH, is buried in its two-chain form (V65 + 10) but it becomes 'exposed' in the presence of glycosaminoglycans (GAGs) e.g. heparin or heparan sulfate. Such a transconformation may be used for targeting the PKA phosphorylation to vitronectin molecules bound to GAGs, for example in the extracellular matrix or on cell surfaces; (f) From the biochemical point of view (Km values and physiological concentrations) the phosphorylation of vitronectin can take place at the locus of a hemostatic event; (g) The phosphorylation of Ser378 in vitronectin alters its function, since it significantly reduces its ability to bind the inhibitor-1 of plasminogen activator(s) (PAI-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

Insulin-like growth factor-binding proteins in R3230AC mammary tumors of intact and diabetic rats.

The insulin-responsive R3230AC mammary tumor possesses type I and type II insulin-like growth factor (IGF) receptors, and membrane preparations display affinity cross-linking of 125I-labeled IGF-I to IGF-binding proteins (IGFBPs). To identify the IGFBPs produced, Northern blotting analysis of poly(A)+ RNA extracts from tumor tissue was performed. Although transcripts of IGFBP-1 were not detected, intense bands were obtained at 1.7 and 6 kilobases (kb) when hybridized with radiolabeled IGFBP-2 and IGFBP-5 cDNA probes, respectively. A 2.6-kb band and a 2.4-kb weaker band were observed after hybridization with IGFBP-3 and IGFBP-4 probes, respectively. When IGFBP-6 cDNA was used, two bands were seen: a higher mol wt band at 6.3 kb and a smaller one at 1.3 kb. Tumors from streptozotocin-induced diabetic rats displayed an increase in the expression of IGFBP-2, and insulin treatment for 3 days normalized the IGFBP-2 mRNA levels. Tumors from diabetic rats displayed no change in IGFBP-3, -4, -5, and -6 mRNA levels from tumors of normoglycemic rats. However, tumors from insulin-treated rats showed significantly higher levels of mRNA for IGFBP-4 and IGFBP-5 than tumors from normoglycemic or diabetic rats. A similar, but less pronounced, pattern of changes in IGFBP-3 mRNA was seen, whereas levels of IGFBP-6 mRNA were unchanged throughout. To identify the cell type producing the mRNAs for these IGFBPs, in situ hybridization of tissue sections was used. Procedures were established that localized three of the five IGFBPs expressed in this tumor tissue. This technique showed that IGFBP-3 mRNA transcripts were observed mainly in endothelial cells of tumor vasculature, although they were also detected in stromal cells, IGFBP-4 was present mainly in tumor stroma cells, and IGFBP-5 mRNA was expressed predominantly in the epithelial cells of this tumor. Expression of IGFBP-5 mRNA transcripts was significantly diminished in primary and long term cultured R3230AC cells grown in alpha-Minimum Essential Medium and 10% fetal bovine serum. Tumors arising from injection of long term cultured cells that were injected into isologous rats contained high amounts of mRNA transcripts for IGFBP-5, suggesting the presence, in vivo, of positive regulators for the expression of this BP. Tumor cells cultured in the presence of insulin displayed a 2.2- to 2.5-fold increase in the expression of IGFBP-5. These findings imply a role for insulin as a regulator of the expression of IGFBP-5 in the R3230AC adenocarcinoma.

Adenocarcinoma↗

PubMed1992

Characterization of insulin-like growth factor receptors in the insulin-responsive R3230AC mammary adenocarcinoma.

To ascertain whether insulin-like growth factors (IGF-1 and IGF-2) affect the estrogen- and insulin-responsive R3230AC mammary carcinoma, studies of IGF-1 and IGF-2 receptors were conducted on primary-culture tumor cells and on membranes purified from whole tumors. By saturation binding analysis, cells in culture displayed one class of IGF-1 binding sites with an affinity constant, Kd, of 5.5 +/- 0.7 x 10(-9) M, whereas in membrane preparations, high and low affinity IGF-1 binding sites, with Kd's of 5 +/- 1.7 x 10(-9) M and 1 +/- 0.4 x 10(-7) M, respectively, were detected. Specificity of binding was demonstrated by 85% displacement of 125I-IGF-1 with 1,000-fold excess unlabeled IGF-1 and 50% displacement with 6.5 x 10(-9) M IGF-1 or 3 microM insulin. Binding sites for IGF-2 were also demonstrable in cultured cells, having a Kd of 7 +/- 0.8 x 10(-10) M, and 50% displacement was obtained with 1.5 x 10(-9) M IGF-2 or 1.5 x 10(-8) M IGF-1. Cross-linking experiments on cultured cells confirmed the presence of IGF-1 and IGF-2 receptors. In purified tumor membranes, IGF binding proteins (M(r) 28,000-32,000) were also detected; their labeling was not displaced by 10(-5) M insulin. In vitro, the tumor cells secrete one or more IGF binding proteins into the medium. Despite the fact that these cells expressed specific IGF receptors, their growth was apparently independent of these growth factors, since neither IGF-1 nor IGF-2 was mitogenic for R3230AC cells in vitro. Nevertheless, IGF-1 caused concentration-related significant increases in the plating efficiency of these cells. Further studies are necessary to determine the functional role of these growth factors, their receptors, and their binding proteins in the biology of this rodent mammary tumor.

Adenocarcinoma↗

PubMed1991

Endogenous cleavage of the Arg-379-Ala-380 bond in vitronectin results in a distinct conformational change which 'buries' Ser-378, its site of phosphorylation by protein kinase A.

Activation of blood platelets by thrombin was previously shown to specifically release protein kinase A, which in human plasma singles out and phosphorylates one protein, identified as vitronectin. This protein is known to be involved in processes that follow platelet stimulation, specifically, in the binding of heparin (interfering with the heparin-mediated inhibition of thrombin and Factor Xa by antithrombin III), in the growth of endothelial cells and in fibrinolysis. This paper shows that phosphorylation of vitronectin by protein kinase A is stoichiometric (approx. 1 mol/mol), that it is targeted to one site (Ser-378) at the C-terminal edge of the heparin-binding domain, and that it distinguishes between the two physiologically occurring forms of vitronectin: the one-chain (75 kDa) form, and the nicked two-chain (65 + 10 kDa) form, held together by an interchain disulphide bridge. Protein kinase A phosphorylates the one-chain form but not the two-chain form, although Ser-378 and the complete recognition sequence of the kinase are still present in the clipped 65 kDa chain. Cleavage of the Arg-379-Ala-380 bond results therefore in a conformationally distinct form of vitronectin in which Ser-378 is 'buried'. This is demonstrated by our finding that Ser-378 is present in the 65 kDa chain of clipped vitronectin but inaccessible to phosphorylation at physiological pH. Upon binding heparin, the phosphorylation site becomes exposed and able to undergo a stoichiometric phosphorylation at physiological pH.

Alanine↗

PubMed1990

The phosphorylation of the two-chain form of vitronectin by protein kinase A is heparin dependent.

In circulating blood, vitronectin occurs in two forms: a single-chain (75 kDa) and an endogenously clipped two-chain form (65 kDa and 10 kDa) held together by a disulfide bridge. The 75 kDa form was previously shown to be phosphorylated at Ser378 by protein kinase A, released by physiologically stimulated platelets. By contrast, at pH 7.5 the two-chain form is not phosphorylated at all. Heparin or heparan sulfate are shown here to modulate the conformation of clipped vitronectin at physiological pH, exposing Ser378 and allowing its stoichiometric phosphorylation by the kinase. At this pH the two-chain form of vitronectin in plasma exhibits a higher affinity for heparin, and behaves as a flexible molecule, which can conformationally respond to heparin and heparan sulfate, effectors involved in vitronectin function.

Amino Acid Sequence↗

PubMed1990

An enzymatic assay for vitronectin based on its selective phosphorylation by protein kinase A.

The catalytic subunit (C) of cAMP-dependent protein kinase selectively phosphorylates vitronectin, a plasma protein that promotes cell adhesion and platelet aggregation, inhibits the inactivation of thrombin by antithrombin III, and participates in complement function. This specific phosphorylation is used here (a) to develop an enzymatic assay for vitronectin (with C and [gamma-32P]ATP) which can be used to identify the vitronectin-containing fractions at each stage of its purification; (b) to radioactively label vitronectin and differentiate between the intact and the nicked form of this protein in structure-function studies; and (c) to identify possible vitronectin-related proteins in the plasma of other animal species.

Animals↗

PubMed1988

Vitronectin is phosphorylated by a cAMP-dependent protein kinase released by activation of human platelets with thrombin.

Activation of freshly isolated human platelets with a physiological stimulant (thrombin) causes them to release a cAMP-dependent protein kinase which specifically phosphorylates one plasma protein (Mr 75000). This protein is immunochemically and biochemically identified as vitronectin (also know as S protein), which was previously implicated in blood clotting, complement function and cell adhesion.

Adenosine Triphosphate↗

PubMed1988

Platelet stimulation releases a cAMP-dependent protein kinase that specifically phosphorylates a plasma protein.

Rabbit serum is shown to contain a cAMP-dependent protein kinase (biochemically characterized as type II) that specifically phosphorylates a 135-kDa endogenous protein. This endogenous phosphorylation can be reproduced with platelet-rich plasma, after stimulation with thrombin, but not with plasma devoid of platelets. Stimulation of isolated platelets ("washed" by gel filtration) with either thrombin or ADP brings about a release of this kinase. The supernatant of these stimulated platelets, which contains the kinase, does not undergo a cAMP-dependent endogenous phosphorylation because it does not contain the 135-kDa protein substrate. On the other hand, plasma devoid of platelets does not contain cAMP-dependent protein kinase. By combining the supernatant of the physiologically stimulated platelets with the plasma devoid of platelets, it is possible to reconstitute the system and to reproduce the specific endogenous phosphorylation of the 135-kDa target substrate. On the basis of the above evidence it is proposed that upon physiological stimulation of platelets, they release into the blood a cAMP-dependent protein kinase in addition to the well-known release of MgATP. This kinase specifically phosphorylates the 135-kDa plasma protein.

Adenosine Diphosphate↗