Potential role of human cytomegalovirus and p53 interaction in coronary restenosis

Science

Volumn 265, Issue 5170, 1994, Pages 391-394

  Speir, Edith ^a   Modali, Rama ^b   Huang, Eng Shang ^c   Leon, Martin B ^d   Shawl, Fayaz ^e   Finkel, Toren ^a   Epstein, Stephen E ^a  

^a NATIONAL INSTITUTES OF HEALTH   (United States)

^b BioServe Biotechnologies   (United States)

^c UNIVERSITY OF NORTH CAROLINA   (United States)

^d WASHINGTON HOSPITAL CENTER   (United States)

^e WASHINGTON ADVENTIST HOSPITAL   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

PROTEIN P53; VIRUS PROTEIN;

ADULT; AGED; ARTICLE; CONTROLLED STUDY; CORONARY ARTERY OBSTRUCTION; CYTOMEGALOVIRUS INFECTION; HISTOPATHOLOGY; HUMAN; HUMAN CYTOMEGALOVIRUS; HUMAN TISSUE; MAJOR CLINICAL STUDY; PRIORITY JOURNAL; PROTEIN LOCALIZATION; RECURRENT DISEASE; TRANSLUMINAL CORONARY ANGIOPLASTY; VASCULAR SMOOTH MUSCLE;

CYTOMEGALOVIRUS; HUMAN HERPESVIRUS 5;

EID: 0028033418     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.8023160     Document Type: Article

References (37)

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30. Fresh atherectomy specimens were obtained in the catheterization laboratory from 60 patients with restenosis. Tissue fragments (1 to 5 mg) were embedded in freezing compound, frozen in isopentane-dry ice, and cut into 10 to 20 sections of 6 μm each; two sections of 50-μm thickness were cut for DNA isolation and PCR [P. H. Gumerlock, U. R. Poonawallee, F. J. Meyers, R. W. D. White, Cancer Res. 51, 1632 (1991)]. Sections were placed on polylysine-coated slides and stored at -80°C until processed for staining. The sections were thawed at 22°C, fixed in -20°C methanol, air-dried for 30 min, rehydrated in phosphate-buffered saline (PBS), and processed as in E. Speir et al. [Circ. Res. 71, 251 (1992)]. The first and second sections were stained with hematoxylin-eosin and Movat pentachrome, respectively. Subsequent sections were stained with the following antibodies to p53: monoclonal PAb 1801, -421, and -240 at 2 μg/ml (Oncogene Science, Uniondale, NY) and polyclonal CM1 at 1:1000 [C. A. Midgley et al., J. Cell Sci. 101, 183 (1992)]. For nonimmune controls we used mouse myeloma protein Mopc21 (Cappel, Durham, NC) and normal rabbit serum (Sigma).
31. Fresh atherectomy specimens were obtained in the catheterization laboratory from 60 patients with restenosis. Tissue fragments (1 to 5 mg) were embedded in freezing compound, frozen in isopentane-dry ice, and cut into 10 to 20 sections of 6 μm each; two sections of 50-μm thickness were cut for DNA isolation and PCR [P. H. Gumerlock, U. R. Poonawallee, F. J. Meyers, R. W. D. White, Cancer Res. 51, 1632 (1991)]. Sections were placed on polylysine-coated slides and stored at -80°C until processed for staining. The sections were thawed at 22°C, fixed in -20°C methanol, air-dried for 30 min, rehydrated in phosphate-buffered saline (PBS), and processed as in E. Speir et al. [Circ. Res. 71, 251 (1992)]. The first and second sections were stained with hematoxylin-eosin and Movat pentachrome, respectively. Subsequent sections were stained with the following antibodies to p53: monoclonal PAb 1801, -421, and -240 at 2 μg/ml (Oncogene Science, Uniondale, NY) and polyclonal CM1 at 1:1000 [C. A. Midgley et al., J. Cell Sci. 101, 183 (1992)]. For nonimmune controls we used mouse myeloma protein Mopc21 (Cappel, Durham, NC) and normal rabbit serum (Sigma).
32. Fresh atherectomy specimens were obtained in the catheterization laboratory from 60 patients with restenosis. Tissue fragments (1 to 5 mg) were embedded in freezing compound, frozen in isopentane-dry ice, and cut into 10 to 20 sections of 6 μm each; two sections of 50-μm thickness were cut for DNA isolation and PCR [P. H. Gumerlock, U. R. Poonawallee, F. J. Meyers, R. W. D. White, Cancer Res. 51, 1632 (1991)]. Sections were placed on polylysine-coated slides and stored at -80°C until processed for staining. The sections were thawed at 22°C, fixed in -20°C methanol, air-dried for 30 min, rehydrated in phosphate-buffered saline (PBS), and processed as in E. Speir et al. [Circ. Res. 71, 251 (1992)]. The first and second sections were stained with hematoxylin-eosin and Movat pentachrome, respectively. Subsequent sections were stained with the following antibodies to p53: monoclonal PAb 1801, -421, and -240 at 2 μg/ml (Oncogene Science, Uniondale, NY) and polyclonal CM1 at 1:1000 [C. A. Midgley et al., J. Cell Sci. 101, 183 (1992)]. For nonimmune controls we used mouse myeloma protein Mopc21 (Cappel, Durham, NC) and normal rabbit serum (Sigma).
33. SMCs (passage 5, grown from explants of a 1-cm segment of human coronary artery obtained at autopsy of an 18-year-old trauma victim, from National Disease Research Interchange, Philadelphia, PA) were seeded at 20,000 cells per well in eight-chamber glass slides (Nunc) and grown in Medium 199, 20% FBS, 1% antibiotic-antimycotic for 72 hours. After removal of the medium, the cells were infected with 50 μl of viral supernatant for 2 hours. The virus suspension was then removed and replaced with 0.4 ml of growth medium. Four wells each of infected or uninfected controls for each time point were analyzed by immunocytochemistry. Cells were fixed, incubated with monoclonal antibodies 6E1 (to IE72) or 12E2 (to IE84) (Vancouver Biotech.) or with p53-specific antibodies 1801 or 421 overnight at 4°C, then reacted with biotinylated goat antibody to mouse IgG and ABC complex (Vector Labs) and with diaminobenzidine [E. Speir et al., in (24)]. Whereas IE72 expression was maximal at 48 hours after infection and then declined, IE84 expression was low at 48 hours but was apparent in 70% of the infected cells at 7 days and in 100% of the cells at 14 days after infection.
34. For indirect immunofluorescence, human coronary SMCs at passage 5 were seeded at 30,000 per square centimeter in eight-well glass chamber slides in Medium 199 and 10% FBS and grown for 48 hours. After three washes with warm PBS, cells were fixed in 1% paraformaldehyde for 2 min, then in 50% acetone-methanol at -10°C for 7 min. Cells were then air-dried, washed in PBS, and blocked with 1% goat serum (Vector Labs) for 20 min. Antibody 12E2 (50 μl), diluted 1:200 in 0.1% crystalline bovine serum albumin (BSA), was added to the wells for 1 hour at 22°C. Cells were washed in warm PBS and the lissamine-rhodamine conjugated goat secondary antibody (Jackson ImmunoResearch, West Grove, PA; diluted in 1% rabbit serum in PBS at 1:50) was added for 30 min at 37°C. Cells were again washed with warm PBS and polyclonal antibody CM1 (Signet, Dedham, MA; diluted 1:200 in 0.1% BSA) was added to the wells for 1 hour at 22°C. Cells were washed in PBS, fluorescein isothiocy-anate-conjugated secondary antibody [diluted in 1% rabbit IgG in PBS (Vector Labs) at 1:50] was added for 30 min, and the cells were washed again. Finally, cells were mounted in 0.1% p-phenylenediamine dissolved in PBS (pH 8), and adjusted with carbonate buffer (pH 9.0).
35. 14C]chloramphenicol and butyryl coenzyme A [T. Finkel, J. Due, E. R. Fearon, C. V. Dang, G. F. Tomaselli, J. Biol. Chem. 268, 5 (1993)].
36. 14C]chloramphenicol and butyryl coenzyme A [T. Finkel, J. Due, E. R. Fearon, C. V. Dang, G. F. Tomaselli, J. Biol. Chem. 268, 5 (1993)].
37. We thank J. Griner, B. Choi, and M. Seddon for technical support; R. Guzman for the balloon-injured rat carotid tissue; W. John Martin for the CMV-positive and CMV-negative DNA; R. Dreyfuss for the photomicrography; A. Gown for HHF35; C. Midgley for CM-1; and A. Levine for plasmids p50-2 and p11-4.