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. 2006 Oct;8(10):807-16.
doi: 10.1593/neo.06379.

Human cytomegalovirus infection alters PC3 prostate carcinoma cell adhesion to endothelial cells and extracellular matrix

Affiliations

Human cytomegalovirus infection alters PC3 prostate carcinoma cell adhesion to endothelial cells and extracellular matrix

Roman A Blaheta et al. Neoplasia. 2006 Oct.

Abstract

The genome and antigens of human cytomegalovirus (HCMV) are frequently found in prostatic carcinoma. However, whether this infection is causative or is an epiphenomenon is not clear. We therefore investigated the ability of HCMV to promote metastatic processes, defined by tumor cell adhesion to the endothelium and extracellular matrix proteins. Experiments were based on the human prostate tumor cell line PC3, either infected with the HCMV strain Hi (HCMV(Hi)) or transfected with cDNA encoding the HCMV-specific immediate early protein IEA1 (UL123) or IEA2 (UL122). HCMV(Hi) upregulated PC3 adhesion to the endothelium and to the extracellular matrix proteins collagen, laminin, and fibronectin. The process was accompanied by enhancement of beta(1)-integrin surface expression, elevated levels of integrin-linked kinase, and phosphorylation of focal adhesion kinase. IEA1 or IEA2 did not modulate PC3 adhesion or beta(1)-integrin expression. Based on this in vitro model, we postulate a direct association between HCMV infection and prostate tumor transmigration, which is not dependent on IEA proteins. Integrin overexpression, combined with the modulation of integrin-dependent signalling, seems to be, at least in part, responsible for a more invasive PC3(Hi) tumor cell phenotype. Elevated levels of c-myc found in IEA1-transfected or IEA2-transfected PC3 cell populations might promote further carcinogenic processes through accelerated cell proliferation.

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Figures

Figure 1
Figure 1
Immunoperoxidase staining of HCMV (HCMVHi)-infected PC3 prostate tumor cells. PC3 cells were infected at an MOI of 1 and were stained against the HCMV-specific 72-kDa immediate early protein UL123. The efficiency of HCMVHi infection was about 30% (original magnification, x 100).
Figure 2
Figure 2
HCMV promotes tumor cell adhesion. Mock-infected controls, HCMVHi-infected cells (MOI = 1 or 0.1), and UV-inactivated PC3 cells were cultured on HUVEC monolayers for 60 minutes. Nonadherent tumor cells were washed off in each sample, and the remaining cells were fixed and counted in five different fields (5 x 0.25 mm2) using a phase-contrast microscope. The upper figure presents PC3Hi, infected for 24 hours; the lower figure is related to PC3Hi, infected for 72 hours. Adhesion capacity is depicted as tumor cell adhesion per square millimeter (mean ± SD; one of seven representative experiments).
Figure 3
Figure 3
The adhesion of prostate tumor cells to extracellular matrix proteins depends on HCMVHi. PC3 cells were added to immobilized fibronectin, laminin, or collagen for 60 minutes. Mock-infected controls, as well as HCMVHi-infected (MOI = 1 or 0.1) or UV-inactivated PC3 cells, were used. Nonadherent tumor cells were washed off in each sample, and the remaining cells were fixed and counted in five different fields (5 x 0.25 mm2) using a phase-contrast microscope. Mean values were calculated from five counts. Specific adhesion capacity (background adhesion on the plastic surface was subtracted from adhesion to matrix proteins) is depicted as counted cells per 0.25 mm2. One of six representative experiments is shown.
Figure 4
Figure 4
β1-Integrin surface expression on HCMVHi-infected versus noninfected PC3 cells. To determine whether integrin modulation was restricted to HCMVHi-infected PC3 cells, cell cultures were double-stained using, on the first step, a monoclonal antibody directed against the HCMV-specific 72-kDa IEA, labeled with FITC. On the second step, PC3 cells were marked with the PE-conjugated monoclonal antibodies anti-CD49b (α2β1), anti-CD49c (α3β1), or anti-CD49d (α4β1). IEA- (PC3Hi-) and IEA+ cells (PC3Hi+) were gated to obtain two distinct cell populations: population I (IEA+) as HCMV-infected cells, and population II (IEA-) as noninfected tumor cells. The integrin expression of both PC3 subtypes was then detected by FACScan analysis [FL-2H(log) channel histogram analysis; 1 x 104 cells/scan]. To evaluate background staining, mouse IgG1-PE was used as isotype control.
Figure 5
Figure 5
Top: Western blot analysis of α4β1 integrin from the proteins of HCMVHi versus mock-infected PC3 cells. Cell lysates were subjected to SDS-PAGE and blotted on the membrane incubated with anti-α4β1 monoclonal antibody. β-Actin served as internal control. The figure shows one of three representative experiments. Bottom: RT-PCR analysis of α4β1 integrin coding mRNA. HCMVHi versus mock-infected PC3 cells were used. RNA was extracted, reverse-transcribed, and submitted to semiquantitative RT-PCR using gene-specific primers, as indicated in the Materials and Methods section. The figure shows one of three representative experiments.
Figure 6
Figure 6
HCMVHi modulates ILK, FAK, and FAKphospho. Mock-infected controls, HCMVHi-infected cells (MOI = 1), and UV-inactivated PC3 were examined by appropriate monoclonal antibodies, as indicated in the Materials and Methods section. β-Actin served as internal control. One of three representative experiments is shown. The x-axis indicates the time after HCMVHi infection.
Figure 7
Figure 7
Adhesion of PC3 to HUVEC or extracellular matrix proteins is integrin-dependent. PC3 cells were preincubated with β1 function-blocking antibodies (clone 6S6) and then added to HUVEC monolayers or immobilized collagen, laminin, or fibronectin. Adherent cells were counted after 60 minutes. The adhesion of cells not treated with monoclonal antibodies was set at 100%. Adhesion blockade diminished the adhesion to HUVEC and extracellular matrix proteins. One of three representative experiments is shown.
Figure 8
Figure 8
Two-channel fluorescence and Western blot analysis of c-myc. Top: HCMVHi-infected PC3 cell cultures were double-stained using, on the first step, a monoclonal antibody directed against the HCMV-specific 72-kDa IEA, labeled with FITC. On the second step, PC3 cells were marked with the PE-conjugated anti-c-myc monoclonal antibody. IEA- (PC3Hi-) and IEA+ cells (PC3Hi+) were gated to obtain two distinct cell populations: population I (IEA+) as HCMV-infected cells, and population II (IEA-) as noninfected tumor cells. The c-myc expression of both PC3 subtypes was then detected by FACScan analysis [FL-2H(log) channel histogram analysis; 1 x 104 cells/scan]. To evaluate background staining, mouse IgG1-PE was used as isotype control. Bottom: Western blot analysis of c-myc from the proteins of HCMVHi (MOI = 1) versus mock-infected PC3 cells. Cell lysates were subjected to SDS-PAGE and blotted on the membrane incubated with c-myc monoclonal antibody. β-Actin served as internal control. The figure shows one of three representative experiments.
Figure 9
Figure 9
c-myc expression depends on the HCMV-specific immediate early protein IEA1. cDNA encoding HCMV IEA1 (UL123) was cloned into the pBS+/- vector and inserted in the expression vector pHM135. cDNA encoding HCMV IEA2 (UL122) was cloned into the pBS+/- vector and inserted in the expression vector pHM134. cDNA encoding HCMV IEA1 and IEA2 was cloned into the pUC18 vector and inserted in the expression vector pHM127. Control cells were transfected with vectors alone. The upper diagram presents a two-channel analysis of c-myc expression of pHM+ versus pHM- PC3 cell populations. Detailed information about cell staining is given in the Materials and Methods section. The lower diagram presents Western blot analysis of c-myc protein expression level. β-Actin served as internal control. The figure shows one of three representative experiments.

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