['Chin,', 'Khew-Voon,', 'Ph.D.', 'respectively,', 'exhibited', 'increased', 'resistance', 'to', 'cisplatin', '(3).', 'We', 'further', 'assessed', 'the', 'resistance', 'of', 'wild-type', 'CHO', 'cells', 'transfected', 'with', 'a', 'cDNA', 'clone', 'for', 'the', 'yeast', 'CAMP-phosphodiesterase', '(PDE)', 'gene', '(10001/PDE)', 'under', 'the', 'control', 'of', 'the', 'murine', 'sarcoma', 'virus', '5°', 'long', 'terminal', 'repeat.', 'The', 'increased', 'PDE', 'expression', 'in', 'the', 'transfectant', 'results', 'in', 'resistance', 'to', 'growth', 'inhibition', 'by', 'cholera', 'toxin', 'and', 'non-hydrolyzable,', 'membrane', 'permeable', 'cAMP', 'analogues.', 'The', '10001/PDE', 'transfectant', 'was', 'also', 'resistant', 'to', 'cisplatin.', 'In', 'addition,', 'transfection', 'of', 'the', 'wild-type', '10001', 'cells', 'with', 'a', 'mouse', 'dominant', 'mutant', 'Ria', 'subunit', 'gene', 'under', 'the', 'influence', 'of', 'the', 'metallothionein', 'gene', 'promoter', '(2)', 'also', 'conferred', 'increased', 'resistance', 'to', 'cisplatin', '(3).', 'These', 'results', 'suggest', 'that', 'functional', 'inactivation', 'of', 'PKA', 'may', 'potentially', 'alter', 'the', 'sensitivity', 'of', 'CHO', 'and', 'Y1', 'cells', 'to', 'cisplatin.', 'However,', 'the', 'kinase', 'mutants', '10260', 'and', '10265', 'that', 'have', 'defective', 'C', 'subunit', 'and', 'decreased', 'PKA', 'activity,', 'displayed', 'similar', 'sensitivity', 'to', 'cisplatin', 'as', 'the', 'wild-type', 'cells', '(3).', 'These', 'results', 'suggest', 'that', 'the', 'increased', 'resistance', 'to', 'cisplatin', 'may', 'be', 'specifically', 'associated', 'with', 'the', 'mutation', 'of', 'the', 'R', 'subunit', 'gene.', 'To', 'assess', 'the', 'mechanisms', 'of', 'cisplatin', 'resistance', 'in', 'these', 'PKA', 'mutants,', 'electrophoretic', 'mobility', 'shift', 'analysis', 'using', 'a', '148', 'bp', 'cisplatin-damaged', 'DNA', 'fragment', 'as', 'probe', 'and', 'nuclear', 'extracts', 'from', 'CHO', 'and', 'Y1', 'cells', 'was', 'performed.', 'Our', 'results', 'showed', 'an', 'increase', 'in', 'recognition', 'and', 'binding', 'of', 'nuclear', 'factor(s)', 'to', 'the', 'damaged', 'DNA', 'by', 'the', '10248', 'and', 'Kin', '8', 'mutants', '(3).', 'Binding', 'was', 'also', 'observed', 'with', 'the', 'wild-type', 'CHO', 'and', 'Y1', 'nuclear', 'extracts', 'but', 'at', 'significantly', 'lower', 'levels.', 'Increased', 'band', 'shift', 'was', 'also', 'detected', 'with', 'the', 'transfectant', '10001/PDE', '(3).', 'The', 'C', 'subunit', 'mutants', '10260', 'and', '10265', 'exhibited', 'band', 'shifts', 'similar', 'to', 'the', 'wild-type', 'cells.', 'To', 'further', 'verify', 'whether', 'the', 'increased', 'binding', 'to', 'damaged', 'DNA', 'by', 'the', 'RI', 'mutants', 'is', 'associated', 'with', 'altered', 'DNA', 'repair,', 'the', 'host', 'cell', 'reactivation', 'assay', 'of', 'damaged', 'plasmid', 'DNA', 'was', 'used', 'to', 'evaluate', 'their', 'repair', 'capacity.', 'We', 'cotransfected', 'CHO', 'and', 'Y1', 'cells', 'and', 'their', 'RI', 'subunit', 'mutant', 'derivatives', 'with', 'reporter', 'plasmids', 'containing', 'the', 'luciferase', 'gene', 'driven', 'by', 'the', '§V40', 'early', 'gene', 'promoter,', 'and', 'ACTINCAT,', 'containing', 'the', 'chloramphenicol', 'acetyltransferase', '(CAT)', 'gene', 'driven', 'by', 'the', 'chicken', 'B-actin', 'gene', 'promoter.', 'The', 'luciferase', 'plasmid', 'was', 'treated', 'with', 'various', 'concentrations', 'of', 'cisplatin', 'for', '18-24', 'hours', 'to', 'induce', 'cisplatin', 'DNA', 'adducts,', 'The', 'ACTINCAT', 'plasmid', 'was', 'cotransfected', 'as', 'an', 'internal', 'control', 'to', 'monitor', 'the', 'transfection', 'efficiency.', 'We', 'observed', 'that', 'luciferase', 'expression', 'in', '10248', 'and', 'Kin', '8', 'mutants', 'was', 'significantly', 'higher', 'than', 'wild-type', 'cells', 'at', 'each', 'concentration', 'of', 'cisplatin', 'treatment', '(3).', 'There', 'was', 'no', 'significant', 'variation', 'in', 'the', 'transfection', 'efficiency', 'between', 'samples', 'as', 'determined', 'from', 'the', 'CAT', 'activity.', 'The', 'increased', 'luciferase', 'expression', 'observed', 'with', 'the', 'Tautants', 'suggests', 'an', 'increased', 'rate', 'of', 'DNA', 'repair.', 'We', 'have', 'also', 'assessed', 'the', 'repair', 'capacity', 'of', 'the', 'mutants', 'in', 'response', 'to', 'UV', 'damage', 'and', 'found', 'the', 'Kin', '8', 'mutant', 'repaired', 'and', 'reactivated', 'the', 'UV-irradiated', 'plasmid', 'more', 'efficiently', 'than', 'wild-type', 'cells.', 'Significance', 'of', 'Work', 'Accomplished', 'by', 'Us:', 'As', 'evident', 'from', 'our', 'results,', 'mutants', 'of', 'the', 'CHO', 'and', 'the', 'adrenocortical', 'Y1', 'cells', 'harboring', 'a', 'defective', 'RI', 'subunit', 'of', 'PKA', 'are', 'resistant', 'to', 'cisplatin', 'as', 'well', 'as', 'other', 'DNA-', 'damaging', 'chemotherapeutic', 'agents.', 'The', 'mechanism', 'of', 'resistance', 'may', 'be', 'associated', 'with', 'increased', 'recognition', 'and', 'binding', 'to', 'cisplatin-', 'and', 'UV-damaged', 'DNA', 'by', 'these', 'mutants.', 'The', 'elevation', 'in', 'band', 'shift', 'is', 'accompanied', 'by', 'increased', 'reactivation', 'of', 'cisplatin-', 'and', 'UV-damaged', 'reporter', 'plasmids', 'in', 'the', 'RJ', 'mutants,', 'suggesting', 'increased', 'DNA', 'repair', 'in', 'the', 'mutants.', 'In', 'addition,', 'overexpression', 'of', 'a', 'dominant', 'mutant', 'Rla', 'subunit', 'gene', 'in', 'the', 'wild-type', 'cells', 'also', 'confers', 'both', 'cAMP-', 'and', 'cisplatin-', 'resistance,', 'further', 'suggesting', 'that', 'RIla', 'may', 'play', 'a', 'direct', 'role', 'in', 'mediating', 'the', 'resistance.', 'As', 'for', 'the', 'observation', 'with', 'the', '10001/PDE', 'cells,', 'which', 'overexpress', 'the', 'cAMP-', 'phosphodiesterase,', 'it', 'seems', 'to', 'be', 'consistent', 'with', 'our', 'hypothesis', 'that', 'the', 'RI', 'subunit', 'may', 'play', 'a', 'role', 'in', 'regulating', 'DNA', 'repair.', 'In', 'our', 'model,', 'the', 'presence', 'of', 'basal', 'or', 'elevated', 'level', 'of', 'cAMP', 'in', 'wild-type', 'cells', 'may', 'promote', 'the', 'binding', 'of', 'cAMP', 'to', 'the', 'free', 'or', 'excess', 'R', 'subunit,', 'which', 'may', 'then', 'associate', 'with', 'and', 'inhibit', 'the', 'constitutively', 'active', 'DNA', 'repair', 'machinery.', 'Overexpression', 'of', 'the', 'cAMP-phosphodiesterase', 'gene,', 'which', 'causes', 'a', 'reduction', 'in', 'the', 'cAMP', 'level,', 'may', 'result', 'in', 'reduced', 'activation', 'of', 'the', 'R', 'subunit', 'and', 'its', 'association', 'with', 'the', 'repair', 'complex.', 'Consequently,', 'the', 'repair', 'machinery', 'may', 'be', 'activated', 'and', 'may', 'lead', 'to', 'an', 'apparent', 'increase', 'in', 'cisplatin', 'resistance.', 'On', 'the', 'other', 'hand,', 'mutation', 'of', 'the', 'RI', 'subunit', 'in', '10248', 'cells', 'may', 'have', 'disrupted', 'its', 'capacity', 'to', 'associate', 'with', 'and', 'negatively', 'regulate', 'DNA', 'repair,', 'thus', 'resulting', 'in', 'an', 'elevated', 'rate', 'of', 'DNA', 'repair.', 'D.', 'Research', 'Design', 'and', 'Methods:', 'Results', 'from', 'our', 'studies', 'suggest', 'that', 'PKA', 'may', 'regulate', 'DNA', 'repair.', 'Therefore,', 'we', 'propose', 'the', 'following', 'experiments', 'to', 'address', 'these', 'issues', 'and', 'in', 'addition,', 'to', 'investigate', 'the', 'opportunity', 'for', 'exploiting', 'cAMP-modulating', 'agents', 'to', 'reverse', 'cisplatin', 'resistance.', ' ', '1.', 'Analysis', 'of', 'the', 'role', 'of', 'R', 'and', 'C', 'subunits', 'of', 'PKA', 'in', 'cisplatin', 'resistance:', 'To', 'demonstrate', 'the', 'role', 'of', 'Rio.', 'in', 'cisplatin', 'resistance', 'unequivocally,', 'we', 'propose', 'to', 'conduct', 'the', 'transfection', 'with', 'the', 'expression', 'vector', 'containing', '50519299']