Tamar Kreizman

Phosphocholine as a biomarker of breast cancer: Molecular and biochemical studies

The discovery of metabolic and molecular markers that help improving the detection and diagnosis of breast cancer is an important goal to be achieved. A high composite‐choline signal in magnetic resonance spectra of breast lesions has been demonstrated to improve the accuracy of breast cancer diagnosis. In the present study we revealed the principal molecular and biochemical steps associated with the induction of choline metabolism and phosphocholine accumulation in human breast cancer cell‐lines in comparison with normal human mammary epithelial cells. We found upregulation of the expression levels of specific choline transporters: organic cation transporter‐2 and choline high affinity transporter‐1, as well as of the enzyme choline kinase α in the cancerous cells in comparison with that in the normal mammary epithelial cells. The expression levels of choline transporter like‐1, organic cation transporter‐1 and choline kinase β were similar in normal and cancerous cells. We further showed that choline transport rates and choline kinase activity indeed increased by several fold in the cancer cells leading to the elevation of phosphocholine. The results strongly suggest that phosphocholine can serve as a biomarker of breast cancer reflecting upregulation of specific choline transporters and choline kinase genes.

Plasmin cleavage of vitronectin Identification of the site and consequent attenuation in binding plasminogen activator inhibitor‐1

Plasmin is shown to specifically cleave vitronectin at the Arg361‐Ser362 bond, 18 amino acid residues upstream from the site of the endogenous cleavage which gives rise to the two‐chain form of vitronectin in plasma. The cleavage site is established using the exclusive phosphorylation of Ser378 with protein kinase A. As a result of the plasmin cleavage, the affinity between vitronectin and the type‐1 inhibitor of plasminogen activator (PAI‐1) is significantly reduced. This cleavage is stimulated by glycosaminoglycans, which are known to anchor vitronectin to the extracellular matrix. A mechanism is proposed through which plasmin can arrest its own production by feedback signalling, unleashing PAI‐1 from the immobilized vitronectin found in the vascular subendothelium, which becomes exposed at the locus of a hemostatic event.

Synthetic peptides derived from the sequence around the plasmin cleavage site in vitronectin: Use in mapping the PAI-1 binding site

A series of 8 peptides derived from the amino acid sequence accommodating the plasmin cleavage site in vitronectin were synthesized and used to map its binding site for the type I plasminogen activator inhibitor (PAI‐1). This mapping assigned the inhibitor binding site to the K348‐R370 region with high affinity recognition elements within the K348‐R357 sequence. These results account for our previous finding that cleavage of the R361 ‐S362 bond by plasmin significantly reduces the affinity between PAI‐1 and vitronectin, since it splits the PAI‐1 binding site in two. Furthermore, in the case of the two‐chain form of vitronectin, this cleavage detaches the S362‐R379 peptide which provides some of the affinity elements for the binding of PAI‐1.

Estrogen regulation of vascular endothelial growth factor in breast cancer in vitro and in vivo: the role of estrogen receptor α and c-Myc

The role of c-Myc in estrogen regulation of vascular endothelial growth factor (VEGF) and of the vasculature function has been investigated in breast cancer cells and tumors. The studies were performed on MCF7 wild-type cells and MCF7-35im clone, stably transfected with an inducible c-Myc gene. In vitro and ex vivo methods for investigating molecular events were integrated with in vivo magnetic resonance imaging of the vascular function. The results showed that the c-Myc upregulation by estrogen is necessary for the transient induction of VEGF transcription; however, overexpression of c-Myc alone is not sufficient for this induction. Furthermore, both c-Myc and the activated estrogen receptor alpha (ERalpha) were shown to co-bind the VEGF promoter in close proximity, indicating a novel mechanism for estrogen regulation of VEGF. Studies of long-term estrogen treatment and overexpression of c-Myc alone demonstrated regulation of stable VEGF expression levels in vitro and in vivo, maintaining steady vascular permeability in tumors. However, withdrawal of estrogen from the tumors resulted in increased VEGF and elevated vascular permeability, presumably due to hypoxic conditions that were found to dominate VEGF overexpression in cultured cells. This work revealed a cooperative role for ERalpha and c-Myc in estrogen regulation of VEGF and the ability of c-Myc to partially mimic estrogen regulation of angiogenesis. It also illuminated the differences in estrogen regulation of VEGF during transient and long-term sustained treatments and under different microenvironmental conditions, providing a complementary picture of the in vitro and in vivo results.

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.

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.

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, Mr 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 pro cesses 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 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). Physiologically, this functional modulation may be involved in ‘unleashing’ PAI-1, allowing its translocation to control the inhibitory function of PAI-1 and, through it, regulating the conversion of plasminogen to active plasmin. (Mol Cell Biochem 127/128: 283–291, 1993)