Yee Sook Cho

Mutations of the gene encoding the protein kinase A type I-α regulatory subunit in patients with the Carney complex

Carney complex (CNC) is a multiple neoplasia syndrome characterized by spotty skin pigmentation, cardiac and other myxomas, endocrine tumours and psammomatous melanotic schwannomas. CNC is inherited as an autosomal dominant trait and the genes responsible have been mapped to 2p16 and 17q22–24 (refs 6, 7). Because of its similarities to the McCune-Albright syndrome and other features, such as paradoxical responses to endocrine signals, genes implicated in cyclic nucleotide-dependent signalling have been considered candidates for causing CNC (ref. 10). In CNC families mapping to 17q, we detected loss of heterozygosity (LOH) in the vicinity of the gene (PRKAR1A) encoding protein kinase A regulatory subunit 1-α (RIα), including a polymorphic site within its 5′ region. We subsequently identified three unrelated kindreds with an identical mutation in the coding region of PRKAR1A. Analysis of additional cases revealed the same mutation in a sporadic case of CNC, and different mutations in three other families, including one with isolated inherited cardiac myxomas. Analysis of PKA activity in CNC tumours demonstrated a decreased basal activity, but an increase in cAMP-stimulated activity compared with non-CNC tumours. We conclude that germline mutations in PRKAR1A, an apparent tumour-suppressor gene, are responsible for the CNC phenotype in a subset of patients with this disease.

Antisense DNAs as multisite genomic modulators identified by DNA microarray

Antisense oligodeoxynucleotides can selectively block disease-causing genes, and cancer genes have been chosen as potential targets for antisense drugs to treat cancer. However, nonspecific side effects have clouded the true antisense mechanism of action and hampered clinical development of antisense therapeutics. Using DNA microarrays, we have conducted a systematic characterization of gene expression in cells exposed to antisense, either exogenously or endogenously. Here, we show that in a sequence-specific manner, antisense targeted to protein kinase A RIα alters expression of the clusters of coordinately expressed genes at a specific stage of cell growth, differentiation, and activation. The genes that define the proliferation-transformation signature are down-regulated, whereas those that define the differentiation-reverse transformation signature are up-regulated in antisense-treated cancer cells and tumors, but not in host livers. In this differentiation signature, the genes showing the highest induction include genes for the G proteins Rap1 and Cdc42. The expression signature induced by the exogenously supplied antisense oligodeoxynucleotide overlaps strikingly with that induced by endogenous antisense gene overexpression. Defining antisense DNAs on the basis of their effects on global gene expression can lead to identification of clinically relevant antisense therapeutics and can identify which molecular and cellular events might be important in complex biological processes, such as cell growth and differentiation.

Protein kinase A isozyme switching: eliciting differential cAMP signaling and tumor reversion

The cAMP-dependent protein kinase types I (PKA-I) and II (PKA-II), composed of identical catalytic (C) subunits but distinct regulatory (R) subunits (RI versus RII), are expressed in a balance of cell growth and differentiation. Distortion of this balance may underlie tumorigenesis and tumor growth. Here, we used PC3M prostate carcinoma cells as a model to overexpress wild type and mutant R and C subunit genes and examined the effects of differential expression of these genes on tumor growth. Only the RIIβ and mutant RIα-P (a functional mimic of RIIβ) transfectants exhibited growth inhibition in vitro, reverted phenotype, and apoptosis, and inhibited in vivo tumor growth. DNA microarrays demonstrated that RIIβ and RIα-P overexpression upregulated a cluster of differentiation genes, while downregulating transformation and proliferation signatures. Overexpression of RIα and Cα, which upregulated PKA-I, elicited the expression signatures opposite that elicited by RIIβ overexpression. Total colocalization of Cα and RIIβ seen by confocal microscopy in the RIIβ cell nucleus supports the opposed genomic regulation demonstrated between Cα and RIIβ cells. Differential expression of PKA R subunits may therefore serve as a tumor-target-based gene therapy for PC3M prostate and other cancers.

Dissecting the Circuitry of Protein Kinase A and cAMP Signaling in Cancer Genesis

Abstract: Expression of the RIα subunit of the cAMP‐dependent protein kinase type I (PKA‐I) is enhanced in human cancer cell lines, in primary tumors, in transformed cells, and in cells upon stimulation of growth. Signaling via the cAMP pathway may be complex, and the biological effects of the pathway in normal cells may depend upon the physiological state of the cells. However, results of different experimental approaches such as antisense exposure, 8‐Cl‐cAMP treatment, and gene overexpression have shown that the inhibition of RIα/PKA‐I exerts antitumor activity in a wide variety of tumor‐derived cell lines examined in vitro and in vivo. cDNA microarrays have further shown that in a sequence‐specific manner, RIα antisense induces alterations in the gene expression profile of cancer cells and tumors. The cluster of genes that define the “proliferation‐transformation” signature are down‐regulated, and those that define the “differentiation‐reverse transformation” signature are up‐regulated in antisense‐treated cancer cells and tumors, but not in host livers, exhibiting the molecular portrait of the reverted (flat) phenotype of tumor cells. These results reveal a remarkable cellular regulation, elicited by the antisense RIα, superimposed on the regulation arising from the Watson‐Crick base‐pairing mechanism of action. Importantly, the blockade of both the PKA and PKC signaling pathways achieved with the CRE‐transcription factor decoy inhibits tumor cell growth without harming normal cell growth. Thus, a complex circuitry of cAMP signaling comprises cAMP growth regulatory function, and deregulation of the effector molecule by this circuitry may underlie cancer genesis and tumor progression.

A genomic-scale view of the cAMP response element-enhancer decoy: A tumor target-based genetic tool

Enhancer DNA decoy oligodeoxynucleotides (ODNs) inhibit transcription by competing for transcription factors. A decoy ODN composed of the cAMP response element (CRE) inhibits CRE-directed gene transcription and tumor growth without affecting normal cell growth. Here, we use DNA microarrays to analyze the global effects of the CRE-decoy ODN in cancer cell lines and in tumors grown in nude mice. The CRE-decoy up-regulates the AP-2β transcription factor gene in tumors but not in the livers of host animals. The up-regulated expression of AP-2β is clustered with the up-regulation of other genes involved in development and cell differentiation. Concomitantly, another cluster of genes involved in cell proliferation and transformation is down-regulated. The observed alterations indicate that CRE-directed transcription favors tumor growth. The CRE-decoy ODN, therefore, may serve as a target-based genetic tool to treat cancer and other diseases in which CRE-directed transcription is abnormally used.

CRE-decoy oligonucleotide-inhibition of gene expression and tumor growth

Nucleic acid molecules with high affinities for a target transcription factor can be introduced into cells as decoy cis-elements to bind these factors and alter gene expression. This review discusses a synthetic single-stranded palindromic oligonucleotide, which self-hybridizes to form a duplex/hairpin and competes with cAMP response element (CRE) enhancers for binding transcription factors. This oligonucleotide inhibits CRE- and Ap-1-directed gene transcription and promotes growth inhibition in vitro and in vivo in a broad spectrum of cancer cells, without adversely affecting normal cell growth. Evidence presented here suggests that the CRE-decoy oligonucleotide can provide a powerful new means of combating cancers, viral diseases, and other pathological conditions by regulating the expression of cAMP-responsive genes.