Jozef Spychala

Tumor-promoting functions of adenosine.

Tumor growth is a multifactorial process that, in addition to mutations leading to dysregulated expression of oncogenes and tumor suppressive genes, requires specific conditions that provide a supportive physiological environment at the primary and metastatic sites of the disease. Adenosine is one of the factors potentially contributing to tumor growth that thus far has not received adequate attention, despite evidence for a broad range of cytoprotective, growth-promoting, and immunosuppressive activities. Adenosine accumulates in solid tumors at high concentrations, and has been shown to stimulate tumor growth and angiogenesis and to inhibit cytokine synthesis, adhesion of immune cells to the endothelial wall, and the function of T-cells, macrophages, and natural killer cells. However, the mechanisms whereby adenosine accumulates in cancer and the specific effects that result from this accumulation are not well understood. This article surveys the available evidence that supports an important role of adenosine in cancer.

Role of Estrogen Receptor in the Regulation of Ecto-5′-Nucleotidase and Adenosine in Breast Cancer

Purpose: The purpose is to understand the expression of ecto-5′-nucleotidase (eN), an adenosine producing enzyme with potential roles in angiogenesis, growth, and immunosuppression, in estrogen receptor (ER)-negative and -positive breast cancer. Experimental Design: We investigated the regulation of eN expression at the mRNA and protein levels by α in a panel of breast cancer cell lines that differ in ER status and invasive and metastatic potential. We also determined rates of adenosine formation in cells with high and low eN expression and in ER+ cells treated with estradiol. Results: ER-negative cells express high eN protein and mRNA levels and produce up to 104-fold more adenosine from AMP and ATP. Estradiol and antiestrogen treatments confirm that eN mRNA and protein expression and adenosine generation are negatively regulated through the ER. Endogenous expression of eN in ER− cells transfected with ERα and phorbol ester-induced eN expression in ER+ cells was strongly suppressed by estradiol, suggesting a dominant function of ER. Finally, an examination of 18 clinical breast cancer samples that were analyzed for both ER status and eN expression by Martin et al. (Cancer Res., 60: 2232–2238, 2000) revealed a significant inverse correlation between ER and eN status. Conclusions: Our results show for the first time that eN is negatively regulated by ERα in dominant fashion and suggests that eN expression and its generation of adenosine may relate to breast cancer progression. Additionally, increased expression of eN in a subset of ER-negative cells may serve as a novel marker for a subset of more aggressive breast carcinoma.

Regulation of the Human Inosine Monophosphate Dehydrogenase Type I Gene UTILIZATION OF ALTERNATIVE PROMOTERS

Catalysis of guanine nucleotide formation from IMP in the de novo purine synthetic pathway is carried out by two isoforms of the enzyme inosine monophosphate dehydrogenase (IMPDH) that are catalytically indistinguishable but are encoded by separate genes. In order to assess the potential for cell type-specific expression of IMPDH activity, we have characterized the IMPDH type I gene and identified three major RNA transcripts that are differentially expressed from three different promoters. A 4.0-kilobase pair (kb) mRNA containing 1.3 kb of 5′-untranslated region is expressed in activated peripheral blood lymphocytes and to a far lesser extent in cultured tumor cell lines. The P1 promoter that regulates the transcription of this mRNA has a high degree of sequence identity to an Alu repetitive sequence. A transcript of 2.7 kb is found in a subset of the tumor cell lines examined, whereas a 2.5-kb mRNA species is universally expressed and is the prevalent mRNA in most cell lines and tissues. The relative strengths of the three promoter regions and the effects of variable extents of 5′-flanking sequence on the P3 promoter differ in Jurkat T, as compared with Raji B lymphoid cell lines, demonstrating a complex cell type-specific transcriptional regulation of IMPDH type I gene expression.

Tissue-specific Regulation of the Ecto-5′-nucleotidase Promoter ROLE OF THE cAMP RESPONSE ELEMENT SITE IN MEDIATING REPRESSION BY THE UPSTREAM REGULATORY REGION

We have isolated the 5′ region of the ecto-5′-nucleotidase (low K m 5′-NT) gene and established that a 969-base pair (bp) fragment confers cell-specific expression of a CAT reporter gene that correlates with the expression of endogenous ecto-5′-NT mRNA and enzymatic activity. A 768-bp upstream negative regulatory region has been identified that conferred lymphocyte-specific negative regulation in a heterologous system with a 244-bp deoxycytidine kinase core promoter. DNase I footprinting identified several protected areas including Sp1, Sp1/AP-2, and cAMP response element (CRE) binding sites within the 201-bp core promoter region and Sp1, NRE-2a, TCF-1/LEF-1, and Sp1/NF-AT binding sites in the upstream regulatory region. Whereas the CRE site was essential in mediating the negative activity of the upstream regulatory region in Jurkat but not in HeLa cells, mutation of the Sp1/AP-2 site decreased promoter activity in both cell lines. Electrophoretic mobility shift assay analysis of proteins binding to the CRE site identified both ATF-1 and ATF-2 in Jurkat cells. Finally, phorbol 12-myristate 13-acetate increased the activity of both the core and the 969-bp promoter fragments, and this increase was abrogated by mutations at the CRE site. In summary, we have identified a tissue-specific regulatory region 5′ of the ecto-5′-NT core promoter that requires the presence of a functional CRE site within the basal promoter for its suppressive activity.

Lipid rafts remodeling in estrogen receptor–negative breast cancer is reversed by histone deacetylase inhibitor

Recently, we have found dramatic overexpression of ecto-5′-nucleotidase (or CD73), a glycosylphosphatidylinositol-anchored component of lipid rafts, in estrogen receptor–negative [ER(−)] breast cancer cell lines and in clinical samples. To find out whether there is a more general shift in expression profile of membrane proteins, we undertook an investigation on the expression of selected membrane and cytoskeletal proteins in aggressive and metastatic breast cancer cells. Our analysis revealed a remarkably uniform shift in expression of a broad range of membrane, cytoskeletal, and signaling proteins in ER(−) cells. A similar change was found in two in vitro models of transition to ER(−) breast cancer: drug-resistant Adr2 and c-Jun-transformed clones of MCF-7 cells. Interestingly, similar expression pattern was observed in normal fibroblasts, suggesting the commonality of membrane determinants of invasive cancer cells with normal mesenchymal phenotype. Because a number of investigated proteins are components of lipid rafts, our results suggest that there is a major remodeling of lipid rafts and underlying cytoskeleton in ER(−) breast cancer. To test whether this broadly defined ER(−) phenotype could be reversed by treatment with differentiating agent, we treated ER(−) cells with trichostatin A, an inhibitor of histone deacetylase, and observed reversal of mesenchymal and reappearance of epithelial markers. Changes in gene and protein expression also included increased capacity to generate adenosine and altered expression profile of adenosine receptors. Thus, our results suggest that during transition to invasive breast cancer there is a significant structural reorganization of lipid rafts and underlying cytoskeleton that is reversed upon histone deacetylase inhibition. [Mol Cancer Ther 2006;5(2):238–45]

Comparative studies on muscle AMP-deaminase—I purification, molecular weight, subunit structure and metal content of the enzymes from rat, rabbit, hen, frog and pikeperch

1. AMP-deaminase (EC 3.5.4.6) from skeletal muscle of frog and pikeperch was purified to homogeneity and compared with the homogeneous enzymes purified from rat, rabbit and hen skeletal muscle. 2. Their molecular weight was close to 280,000, every enzyme consisted of four identical subunits of molecular weight about 70,000. 3. All enzymes were found to contain about two atoms of zinc per molecule. 4. Minor differences of u.v.-absorption spectra between amphibian and fish muscle enzyme as compared with mammalian and bird muscle enzyme were found.

Ecto-5′-Nucleotidase (eN, CD73) is Coexpressed with Metastasis Promoting Antigens in Human Melanoma Cells

Upregulated expression of eN has been found in the highly invasive human melanoma cell lines but neither in melanocytes nor in primary tumor cells. Membrane proteins associated with cell adhesion and metastasis: α5-, β1-, β3-integrins, and CD44 were elevated gradually in accordance with increasing metastatic potential. αv-integrin was seen mostly in aggressive melanomas. The expression of eN correlated with a number of metastasis-related markers and thus may have a function in the process. eN activity went parallel with its amount in all cells. Concanavalin A strongly inhibited the enzyme in a noncompetitive way. Clustering of eN protein in overexpressing cells by ConA-treatment increased the enzyme association with the heavy cytoskeletal complexes. A similar shift towards cytoskeletal fractions took also place with other membrane proteins coexpressed with eN. This ConA-induced association may reflect a putative interaction of eN with physiological ligand, that upon interaction, aggregates protein components of lipid rafts and triggers signaling pathway that may be intrinsically involved in cell-stroma adhesion.

Comparative studies on muscle AMP-deaminase--II. Regulation by monovalent cations, ATP and orthophosphate of the enzyme from hen, frog and pikeperch muscle.

1. Michaelis constants, maximum velocity and pH-dependence of the reaction catalysed by homogeneous AMP-deaminase preparations from hen, frog and pikeperch skeletal muscle were compared, as well as the influence of monovalent cations, ATP and inorganic phosphate. 2. ATP was found to activate the enzymes in the absence of K+ and at optimum (150 mM) KCl concentration. 3. Absolute dependence on potassium ions and considerable dependence of Km and Vmax on the kind of monovalent cation present in the medium were found for pikeperch enzyme.

Enforced expression of cytosolic 5'-nucleotidase I confers resistance to nucleoside analogues in vitro but systemic chemotherapy toxicity precludes in vivo selection

Purpose: Retroviral transfer of cDNA sequences that confer drug resistance can be used to protect against chemotherapy-induced hematopoietic toxicity and for the selective expansion of gene-modified cells. To successfully expand genetically engineered cells in vivo, an appropriate balance must be achieved between systemic toxicity induced by the selecting agent and the expansion of modified cells. Method: In this study, we investigate retroviral transfer of cytosolic 5′-nucleotidase I (cN-I) for protection and selection of gene-modified cells when treated with 2-chloro-2′-deoxyadenosine (2-CdA) and 5-fluorouracil (5-FU) alone and in combination. We also attempt to design a treatment strategy for the potential in vivo selection of cN-I-modified cells by administering 5-FU to mice prior to 2-CdA treatment. Results: Our results show that cN-I can be transferred by recombinant retroviruses, and that enforced expression of cN-I protects murine fibroblast and hematopoietic progenitor cells from the cytotoxic effects of 2-CdA and/or 5-FU. Furthermore, we show that the combined administration of 5-FU and 2-CdA potentiates hematopoietic stem cell toxicity. However, the treatment also results in severe myelosuppression. Conclusion: These results show that while cN-I provides both protective and selective benefits to gene-modified cells in vitro, selection requires a treatment strategy that is likely too toxic to consider cN-I as an in vivo selectable marker

Regulation of Low Km (Ecto) 5’-Nucleotidase Gene Expression in Leukemic Cells

Ecto-5’-NT has been considered a biochemical marker in leukemias and lymphomas (1). The enzyme activity is low in lymphocytes (2) and decreased in B-cells in patients with X-linked agammaglobulinemia (3), decreased or not detectable in lymphocytes from chronic lymphocytic leukemia and infectious mononucleosis (4) and decreased in CD8+ lymphocytes in AIDS (5). In general, low activity has been observed during development and in undifferentiated cells which increases markedly in some cell types at the final stages of differentiation and during senescence and aging. Although there are some data showing correlation of low ecto-5’-NT activity with efficacy of nucleoside analogues of anti-cancer activity, no specific role which would explain the pattern of distribution of this enzyme has been proposed.