Concurrent Expression of Hyaluronan Biosynthetic and Processing Enzymes Promotes Growth and Vascularization of Prostate Tumors in Mice
2006; Elsevier BV; Volume: 169; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2006.060032
ISSN1525-2191
Autores Tópico(s)Glycosylation and Glycoproteins Research
ResumoAggressive cells in prostate cancer secrete extracellular hyaluronan (HA) as a result of up-regulated HA synthase enzymes HAS2 and HAS3. Combined detection of HA and the HA processing hyaluronidase enzyme Hyal1 in prostate tumors correlates with poor outcome. HA oligomers produced by hyaluronidases are potent angiogenic stimuli. We investigated the respective roles of HAS2 and Hyal1 using 22Rv1 human prostate tumor cells that lack both enzyme activities. Stable transfectants were selected for overexpression of Hyal1 or HAS2 and for coexpression of Hyal1 and HAS2. HAS2 overexpression elevated HA production and excess pericellular HA retention. However, HAS2-transfected tumor cell growth in culture was dramatically slowed. Coexpression of Hyal1 with HAS2 diminished HA retention but restored growth kinetics, supporting a possible combined role for excess HA synthesis and processing in maximizing unrestricted growth of prostate cancer cells. In mice, overexpression of HAS2 increased subcutaneous tumor size. Excess activity of either Hyal1 or HAS2 enhanced angiogenesis, but the most significant tumorigenic potential was realized by coexpression of both Hyal1 and HAS2 enzymes. Thus, HA production by tumor cells in prostate cancer may enhance the aggressive potential of the cells by increasing Hyal1-dependent autocrine proliferation and potentiating vascular development. Aggressive cells in prostate cancer secrete extracellular hyaluronan (HA) as a result of up-regulated HA synthase enzymes HAS2 and HAS3. Combined detection of HA and the HA processing hyaluronidase enzyme Hyal1 in prostate tumors correlates with poor outcome. HA oligomers produced by hyaluronidases are potent angiogenic stimuli. We investigated the respective roles of HAS2 and Hyal1 using 22Rv1 human prostate tumor cells that lack both enzyme activities. Stable transfectants were selected for overexpression of Hyal1 or HAS2 and for coexpression of Hyal1 and HAS2. HAS2 overexpression elevated HA production and excess pericellular HA retention. However, HAS2-transfected tumor cell growth in culture was dramatically slowed. Coexpression of Hyal1 with HAS2 diminished HA retention but restored growth kinetics, supporting a possible combined role for excess HA synthesis and processing in maximizing unrestricted growth of prostate cancer cells. In mice, overexpression of HAS2 increased subcutaneous tumor size. Excess activity of either Hyal1 or HAS2 enhanced angiogenesis, but the most significant tumorigenic potential was realized by coexpression of both Hyal1 and HAS2 enzymes. Thus, HA production by tumor cells in prostate cancer may enhance the aggressive potential of the cells by increasing Hyal1-dependent autocrine proliferation and potentiating vascular development. Prostate cancer is the second leading cause of cancer death in men.1Landis SH Murray T Bolden S Wingo PA Cancer statistics, 1999.CA Cancer J Clin. 1999; 49: 8-31Crossref PubMed Scopus (3135) Google Scholar Although treatment efficacy has improved dramatically by increased awareness and earlier diagnosis, a large number of cases progress to aggressive, metastatic disease. Understanding the molecular phenomena that lead to progression would improve diagnostic, prognostic, and therapeutic indices. A growing body of evidence suggests that aggressive progression of cancer is facilitated by changes in the composition of extracellular matrix surrounding the cancerous tissue. One matrix molecule implicated in the progression of prostate cancer is hyaluronan (HA), a large glycosaminoglycan polymer that is not prominent in normal tissue or benign prostatic hyperplasia but is abundant in tissue biopsies from patients in various stages of prostate cancer.2Aaltomaa S Lipponen P Tammi R Tammi M Viitanen J Kankkunen JP Kosma VM Strong stromal hyaluronan expression is associated with PSA recurrence in local prostate cancer.Urol Int. 2002; 69: 266-272Crossref PubMed Scopus (70) Google Scholar, 3De Klerk DP The glycosaminoglycans of normal and hyperplastic prostate.Prostate. 1983; 4: 73-81Crossref PubMed Scopus (27) Google Scholar, 4De Klerk DP Lee DV Human HJ Glycosaminoglycans of human prostatic cancer.J Urol. 1984; 131: 1008-1012Crossref PubMed Scopus (48) Google Scholar, 5Ekici S Cerwinka WH Duncan R Gomez P Civantos F Soloway MS Lokeshwar VB Comparison of the prognostic potential of hyaluronic acid, hyaluronidase (HYAL-1), CD44v6 and microvessel density for prostate cancer.Int J Cancer. 2004; 112: 121-129Crossref PubMed Scopus (81) Google Scholar, 6Lokeshwar VB Rubinowicz D Schroeder GL Forgacs E Minna JD Block NL Nadji M Lokeshwar BL Stromal and epithelial expression of tumor markers hyaluronic acid and HYAL1 hyaluronidase in prostate cancer.J Biol Chem. 2001; 276: 11922-11932Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 7Posey JT Soloway MS Ekici S Sofer M Civantos F Duncan RC Lokeshwar VB Evaluation of the prognostic potential of hyaluronic acid and hyaluronidase (HYAL1) for prostate cancer.Cancer Res. 2003; 63: 2638-2644PubMed Google Scholar Normally, HA is produced during specific events that require cellular proliferation and motility, such as developmental neural crest cell migration,8Fujimoto T Hata J Yokoyama S Mitomi T A study of the extracellular matrix protein as the migration pathway of neural crest cells in the gut: analysis in human embryos with special reference to the pathogenesis of Hirschsprung's disease.J Pediatr Surg. 1989; 24: 550-556Abstract Full Text PDF PubMed Scopus (63) Google Scholar cardiac morphogenesis,9Camenisch TD Spicer AP Brehm-Gibson T Biesterfeldt J Augustine ML Calabro Jr, A Kubalak S Klewer SE McDonald JA Disruption of hyaluronan synthase-2 abrogates normal cardiac morphogenesis and hyaluronan-mediated transformation of epithelium to mesenchyme.J Clin Invest. 2000; 106: 349-360Crossref PubMed Scopus (719) Google Scholar prostate development,10Gakunga P Frost G Shuster S Cunha G Formby B Stern R Hyaluronan is a prerequisite for ductal branching morphogenesis.Development. 1997; 124: 3987-3997Crossref PubMed Google Scholar and wound healing.11Yung S Thomas GJ Davies M Induction of hyaluronan metabolism after mechanical injury of human peritoneal mesothelial cells in vitro.Kidney Int. 2000; 58: 1953-1962Abstract Full Text Full Text PDF PubMed Google Scholar However, its accumulation is tightly controlled and restricted in adulthood to extracellular matrices within joint tissues, vitreous humor, skin, and protective encapsulation of vital organs.12Fraser JR Laurent TC Laurent UB Hyaluronan: its nature, distribution, functions and turnover.J Intern Med. 1997; 242: 27-33Crossref PubMed Scopus (1512) Google Scholar, 13Laurent TC Laurent UB Fraser JR The structure and function of hyaluronan: an overview.Immunol Cell Biol. 1996; 74: A1-A7Crossref PubMed Scopus (401) Google Scholar Steady-state levels of HA are maintained by the cumulative efforts of two families of enzymes: the HA synthases14Weigel PH Hascall VC Tammi M Hyaluronan synthases.J Biol Chem. 1997; 272: 13997-14000Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar (HAS1, HAS2, and HAS3 in mammals) and the hyaluronidases15Csoka AB Frost GI Stern R The six hyaluronidase-like genes in the human and mouse genomes.Matrix Biol. 2001; 20: 499-508Crossref PubMed Scopus (487) Google Scholar (Hyal1 through Hyal4 and PH20). HA synthases are integral membrane enzymes that synthesize HA polymers of 105 to 107 d, simultaneously secreting them to the extracellular space as they are polymerized. 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Quantitative measurement of HA accumulation in human prostate tumors by histopathological analysis, in combination with elevated expression of the hyaluronidase Hyal1, is an independent indicator of poor prognosis for patients.5Ekici S Cerwinka WH Duncan R Gomez P Civantos F Soloway MS Lokeshwar VB Comparison of the prognostic potential of hyaluronic acid, hyaluronidase (HYAL-1), CD44v6 and microvessel density for prostate cancer.Int J Cancer. 2004; 112: 121-129Crossref PubMed Scopus (81) Google Scholar, 6Lokeshwar VB Rubinowicz D Schroeder GL Forgacs E Minna JD Block NL Nadji M Lokeshwar BL Stromal and epithelial expression of tumor markers hyaluronic acid and HYAL1 hyaluronidase in prostate cancer.J Biol Chem. 2001; 276: 11922-11932Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 7Posey JT Soloway MS Ekici S Sofer M Civantos F Duncan RC Lokeshwar VB Evaluation of the prognostic potential of hyaluronic acid and hyaluronidase (HYAL1) for prostate cancer.Cancer Res. 2003; 63: 2638-2644PubMed Google Scholar This correlative finding is consistent with studies that show an active role for HA in tumor progression. 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Hyal1 converts polymeric HA to oligomeric species, some of which have potent biological function. 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Previous studies with human prostate tumor cell lines have shown that excess HA production correlates with aggressive potential of the cell.33Simpson MA Reiland J Burger SR Furcht LT Spicer AP Oegema Jr, TR McCarthy JB Hyaluronan synthase elevation in metastatic prostate carcinoma cells correlates with hyaluronan surface retention, a prerequisite for rapid adhesion to bone marrow endothelial cells.J Biol Chem. 2001; 276: 17949-17957Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar HAS2 and HAS3 isozymes were found to be specifically up-regulated in the aggressive cells. Inhibition of HAS2 or HAS3 in these cells significantly impairs tumor growth and spontaneous metastatic potential.34Simpson MA Wilson CM Furcht LT Spicer AP Oegema Jr, TR McCarthy JB Manipulation of hyaluronan synthase expression in prostate adenocarcinoma cells alters pericellular matrix retention and adhesion to bone marrow endothelial cells.J Biol Chem. 2002; 277: 10050-10057Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 35Simpson MA Wilson CM McCarthy JB Inhibition of prostate tumor cell hyaluronan synthesis impairs subcutaneous growth and vascularization in immunocompromised mice.Am J Pathol. 2002; 161: 849-857Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar Not surprisingly, HA-deficient tumors are very poorly vascularized relative to HA-rich tumors. However, the respective and concerted roles of hyaluronidase and HA synthase in HA accumulation and utilization by tumor cells have not been examined. In this report, we have selected and characterized prostate tumor cell transfectants for expression of HAS2 or Hyal1 individually and for coexpression of Hyal1/HAS2. Cell lines were compared for enzyme activity, growth properties in vitro and in vivo, and angiogenic potential in mice. Excess HA production increased cell-surface HA retention and slowed cellular growth dramatically. Interestingly, coexpression of Hyal1 virtually eliminated pericellular HA retention and restored growth rates to control levels. The cotransfected cells exhibited dramatic increases in tumor growth rate on subcutaneous injection. Angiogenesis was stimulated significantly by all three transfection conditions but did not correlate with tumorigenic potential of the cells. This is the first report to begin systematically assessing the roles of HA biosynthesis and degradation in prostate tumor cell biology. The results are consistent with an active role of both processes in progression of prostate cancer. The PC3 derivative cell line, PC3M-LN4 (human prostate adenocarcinoma cells), was kindly provided by Dr. Isaiah J. Fidler (M.D. Anderson Hospital Cancer Center, Houston, TX) and cultured similarly to PC3 in minimal essential medium supplemented with 10% fetal bovine serum, nonessential amino acids, and 1 mmol/L sodium pyruvate. 22Rv1 and LNCaP cells were purchased from the American Type Culture Collection (Manassas, VA) and cultured in Roswell Park Memorial Institute (RPMI) 1640/10% fetal bovine serum as recommended. Biotinylated HA-binding protein (HABP) was from Seikagaku (Northstar BioProducts, Fast Falmouth, MA). HA used in the quantification assay was purchased from Sigma-Aldrich (St. Louis, MO). Anti-mouse CD31-phycoerythrin conjugate was from BD Biosciences (San Diego, CA). Expression of HAS isozymes 1, 2, and 3 was assayed by reverse transcriptase-polymerase chain reaction (RT-PCR) as previously described.33Simpson MA Reiland J Burger SR Furcht LT Spicer AP Oegema Jr, TR McCarthy JB Hyaluronan synthase elevation in metastatic prostate carcinoma cells correlates with hyaluronan surface retention, a prerequisite for rapid adhesion to bone marrow endothelial cells.J Biol Chem. 2001; 276: 17949-17957Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar Hyal1 in prostate carcinoma cell lines was assayed similarly. Briefly, poly(A)+ RNA was isolated from subconfluent tumor cells (Oligotex mRNA isolation kit; Qiagen, Valencia, CA) and quantified, and equal amounts were reverse-transcribed with an oligo(dT) primer using the Superscript III kit (Invitrogen, Carlsbad, CA). Polymerase chain reaction (PCR) oligonucleotides were designed using the published sequences. Target amplification sequences of 200–400 bases were selected and amplified in 25–35 cycles of 30-seconds denaturation at 95°C, 30-seconds annealing at 60°C, and 30-seconds extension at 72°C. Glyceraldehyde-3-phosphate dehydrogenase was amplified in identical simultaneous reactions as a normalization control. HAS2 expression in stable transfectants was similarly assayed. The plasmid construct encoding HAS2 was previously described.34Simpson MA Wilson CM Furcht LT Spicer AP Oegema Jr, TR McCarthy JB Manipulation of hyaluronan synthase expression in prostate adenocarcinoma cells alters pericellular matrix retention and adhesion to bone marrow endothelial cells.J Biol Chem. 2002; 277: 10050-10057Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar The Hyal1 coding sequence (accession U96078) was amplified by RT-PCR from PC3M-LN4 cells, subcloned with a Flag epitope tag, and placed in the pIRES2-EGFP bicistronic expression vector (Clontech, Palo Alto, CA) for constitutive expression and antibiotic selection in eukaryotic cells. The sequence was verified for identity to the published sequence. Expression of each coding sequence was confirmed as described below. 22Rv1 human prostate tumor cells were transfected with 1) vector alone (pIRES2-EGFP); 2) plasmid encoding Hyal1-Flag; 3) HAS2 plasmid; or 4) both the Hyal1 and the HAS2 plasmid constructs. Transfections were performed via a liposome-mediated method using FuGene6 reagent (Roche Applied Science, Mannheim, Germany), according to the manufacturer's protocol. Gene expression was verified by RT-PCR (HAS2) or Western blot (Hyal1) and by enzyme activity assays as described. Stable selection of 22Rv1 transfectants was by clonal isolation with cloning disks following incubation for 14 to 20 days with G418 (1.25 mg/ml). Multiple clones were tested for expression, and those with average levels of expression were pooled to eliminate bias from isolated selection. Once established, stable transfectants were maintained in the selection medium. Cell culture supernatants from transfected cell lines were concentrated approximately 10-fold and electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 12% polyacrylamide gel containing 0.2 mg/ml HA. The gel was soaked for 1 hour at room temperature in 3% Triton X-100 and then incubated in hyaluronidase assay buffer (50 mmol/L sodium formate, pH 4.0, 150 mmol/L NaCl, 0.1% bovine serum albumin (BSA)) at 37°C overnight. Hyaluronidase activity was detected as a clear band at the specific molecular weight (eg, 58 kd) for Hyal1 on staining 1 hour with 0.5% Alcian blue and destaining with 7% acetic acid. Cell lysates were assayed similarly, but, although several light bands were visible, no significant differences were observed between control and Hyal1-transfected cells. Thus, the majority of overexpressed Hyal1 was secreted. To determine whether supernatants or whole cell lysates contained differential activities of the pH neutral hyaluronidases (Hyal2 or PH20), substrate gels generated as above were also assayed in 50 mmol/L sodium phosphate, pH 7.0, containing 150 mmol/L NaCl and 0.1% BSA. No activity was observed in the conditioned media, and, again, several faint bands were present in lysates without significant differences between control and transfected cells. Furthermore, the pattern was not appreciably different between lysates assayed at pH 7 versus pH 4. Hence, these data are not shown. The concentration of HA in transfected cell culture supernatants was determined in a competitive binding assay.36Frost GI Stern R A microtiter-based assay for hyaluronidase activity not requiring specialized reagents.Anal Biochem. 1997; 251: 263-269Crossref PubMed Scopus (123) Google Scholar Briefly, Immulon 96-well microtiter plates were coated with human umbilical cord HA at 50 μg/ml in 200 mmol/L carbonate buffer (pH 9.6) overnight at 4°C. Excess HA was removed, and wells were blocked with Superblock reagent (Pierce, Rockford, IL). Overnight conditioned culture media from prostate tumor cell cultures were harvested, and cell counts were determined manually. Cell culture supernatants were centrifuged, boiled 20 minutes, and serially diluted in phosphate-buffered saline/0.05% Tween 20. Equal volumes of each dilution were combined with biotinylated HABP to a final HABP concentration of 0.5 μg/ml and incubated in the HA-precoated wells at room temperature for 6 to 8 hours. Plates were washed four times with phosphate-buffered saline/Tween 20 and developed using an avidin-biotin horseradish peroxidase system (ABC-HRP kit PK-4000; Vector Laboratories, Burlingame, CA) with tetramethylbenzidine (Sigma-Aldrich) as substrate, and absorbance was read at 650 nm in a PowerWave microplate spectrophotometer (BioTek, Winooski, VT). HA concentration was interpolated from a standard curve of absorbance values corresponding to known HA quantities. The mean HA concentration for each sample of culture supernatant was calculated, and results were normalized to cell number. Pericellular HA retention was visualized and quantified by particle exclusion as described previously.34Simpson MA Wilson CM Furcht LT Spicer AP Oegema Jr, TR McCarthy JB Manipulation of hyaluronan synthase expression in prostate adenocarcinoma cells alters pericellular matrix retention and adhesion to bone marrow endothelial cells.J Biol Chem. 2002; 277: 10050-10057Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 35Simpson MA Wilson CM McCarthy JB Inhibition of prostate tumor cell hyaluronan synthesis impairs subcutaneous growth and vascularization in immunocompromised mice.Am J Pathol. 2002; 161: 849-857Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar Briefly, prostate carcinoma cells cultured overnight in 48-well plates were washed and incubated 90 minutes with 2 mg/ml aggrecan in phenol red-free RPMI with 0.1% BSA at 37°C. The aggrecan solution was removed, and 1 × 108 glutaraldehyde-fixed sheep red blood cells (Accurate Chemical and Scientific Corp., Westbury, NY) in phosphate-buffered saline/1% BSA were added, allowed to settle for 15 minutes, and then viewed with phase-contrast microscopy. The HA matrix was evidenced by halos surrounding the cells from which the fixed erythrocytes were excluded. Representative cells were photographed at magnification ×400. To quantify matrix retention, outlines of matrices and cellular boundaries from individual cells of each type were traced, and relative areas were calculated using Adobe Photoshop (Adobe Systems Incorporated, San Jose, CA). HA matrix thickness is presented as the ratio of matrix area to cell area for each transfectant or condition, with the mean ratio represented by a horizontal bar. Two-dimensional growth in culture was assayed as previously described.35Simpson MA Wilson CM McCarthy JB Inhibition of prostate tumor cell hyaluronan synthesis impairs subcutaneous growth and vascularization in immunocompromised mice.Am J Pathol. 2002; 161: 849-857Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar Equivalent passages of each tumor cell line were plated at 5000 cells/well in 24-well plates. After 7 days, the most rapidly growing cell line had reached ∼90% confluence, so this was chosen as the experimental endpoint. At 24-hour intervals up to 7 days, quadruplicate wells of each cell line were released with trypsin, neutralized, and manually counted in a hemacytometer. Duplicate counts for each well were averaged to obtain the total cell count per well. Each point is the mean ± SE of the total cell counts. Although the graph presents data from a single assay in quadruplicate, the growth trends were reproduced in three additional identical assays. All mice were cared for and maintained under the supervision and guidelines of the University of NebraskaLincoln Institutional Animal Care and Use Committee. Male non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice (Jackson Laboratories, Bar Harbor, ME, a total of eight animals per condition) were injected subcutaneously in each flank with 1 × 106 tumor cells suspended in 100 μl of serum-free RPMI. Tumor growth was monitored weekly with caliper measurements. After 28 days, mice were sacrificed, and tumors were dissected and weighed. Mean tumor wet weight ± SE was plotted for each cell line. Statistical significance was assigned by Student's two-tailed t-test. The experiment was repeated with an additional eight animals per cell line, and similar results were obtained. HA content and vascularization of tumors were detected as described previously.35Simpson MA Wilson CM McCarthy JB Inhibition of prostate tumor cell hyaluronan synthesis impairs subcutaneous growth and vascularization in immunocompromised mice.Am J Pathol. 2002; 161: 849-857Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar Briefly, after weighing, tumors were divided in halves. One half was formalin-fixed and embedded in paraffin, and the other half was snap frozen in OCT compound. For HA detection, paraffin-embedded tumors were sectioned, dewaxed, incubated with 3 μg/ml biotinylated HABP in phosphate-buffered saline/1% BSA overnight at 4°C, and developed using the Vectastain ABC kit (Vector Laboratories). Sections were counterstained with Meyer's hematoxylin to visualize cellular boundaries. White light images were collected at magnification ×200 on a Leica DM-IRB inverted microscope equipped with an Optronics Magnafire digital camera. Vascularization of the tumors was assessed in acetone-fixed frozen sections (8-μm thickness) by antibody staining for CD31 as described previously.35Simpson MA Wilson CM McCarthy JB Inhibition of prostate tumor cell hyaluronan synthesis impairs subcutaneous growth and vascularization in immunocompromised mice.Am J Pathol. 2002; 161: 849-857Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 37Wild R Ramakrishnan S Sedgewick J Griffioen AW Quantitative assessment of angiogenesis and tumor vessel architecture by computer-assisted digital image analysis: effects of VEGF-toxin conjugate on tumor microvessel density.Microvasc Res. 2000; 59: 368-376Crossref PubMed Scopus (125) Google Scholar CD31-phycoerythrin-conjugated antibody staining of frozen sections was visualized by fluorescence microscopy. Five random sections from each of three tumors per cell line were digitally photographed with 5-s exposure time, saved as TIF files, and processed. Images were converted from 16 to 8 bit in Adobe Photoshop, red channel fluorescence was specifically isolated, images were converted to grayscale and inverted, and a black-and-white threshold was arbitrarily set based on levels. The histogram function was then used to determine vessel density as represented by density of black pixels at 0 on the black-to-white scale. Average pixel density for each transfectant tumor section was normalized to the average pixel density for untransfected tumor sections. Statistical significance was assigned by Student's two-tailed t-test. To assess the relative and concerted roles of HA biosynthesis and processing, tumor cell lines were first characterized for expression of HA synthase and hyaluronidase isozymes. Hyaluronidases have been reported in aggressive prostate cancer cells,6Lokeshwar VB Rubinowicz D Schroeder GL Forgacs E Minna JD Block NL Nadji M Lokeshwar BL Stromal and epithelial expression of tumor markers hyaluronic acid and HYAL1 hyaluronidase in prostate cancer.J Biol Chem. 2001; 276: 11922-11932Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 38Lokeshwar VB Lokeshwar BL Pham HT Block NL Association of ele
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