Strong evidence has indicated that protein phosphatase 2A (PP2A) is a tumor suppressor, but a mouse model for testing the tumor suppressor activity was missing. The most abundant forms of trimeric PP2A holoenzyme consist of the scaffolding A␣ subunit, one of several regulatory B subunits, and the catalytic C␣ subunit. A␣ mutations were discovered in a variety of human carcinomas. All carcinoma-associated mutant A␣ subunits are defective in binding the B or B and C subunits. Here we describe two knock-in mice expressing cancer-associated A␣ point mutants defective in binding B subunits, one knockout mouse expressing truncated A␣ defective in B and C subunit binding, and a floxed mouse for generating conditional A␣ knockouts. We found that the cancer-associated A␣ mutations increased the incidence of cancer by 50 to 60% in lungs of FVB mice treated with benzopyrene, demonstrating that PP2A acts as a tumor suppressor. We show that the effect of A␣ mutation on cancer incidence is dependent on the tumor suppressor p53. The finding that the A␣ mutation E64D, which was detected in a human lung carcinoma, increases the lung cancer incidence in mice suggests that this mutation also played a role in the development of the carcinoma in which it was discovered.Protein phosphatase 2A (PP2A) plays a role in many fundamental cellular processes, such as signal transduction, DNA repair, transcription, translation, and growth control (15,24,26,43,77). The basis for this multifunctionality of PP2A rests on the large number of subunits that control its phosphatase activity, substrate specificity, and subcellular localization. The trimeric holoenzyme is composed of a catalytic C subunit, a scaffolding A subunit, and one of many regulatory B subunits. The dimeric core enzyme consists of one A and one C subunit. Both forms coexist in cultured cells and in tissue (5,30,42). The A subunit exists as two isoforms, A␣ and A␤, which are 87% identical. The catalytic C subunit also exists as two isoforms, C␣ and C␤, which are 96% identical. The B subunits fall into four families designated B, BЈ, BЉ, and Bٞ. The B or PR55 family has four members, the BЈ family (also designated B56 or PR61) consists of five isoforms and additional splice variants, and the BЉ or PR72 family has four members, including splice variants. B, BЈ, and BЉ are largely unrelated by sequence except for two common regions involved in binding to the A subunit (34). The Bٞ family has two members, striatin and S/G 2 nuclear autoantigen (40). The combination of all subunits could give rise to over 70 different holoenzymes. In addition, the ability of PP2A to associate with approximately 150 other proteins further increases its regulatory potential (15,24,26,42). Figure 1 shows a schematic diagram of the BЈ-containing holoenzyme whose structure has been revealed by biochemical analysis (53, 56) and X-ray crystallography (9,19,44,78,79).The first indication that PP2A might be a tumor suppressor came from the finding that okadaic acid is both a tumor promoter and an inhibitor of...

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Alkoxyalkyl esters of cidofovir (CDV) are orally active agents which inhibit the replication of a variety of double stranded DNA (dsDNA) viruses including variola, vaccinia, ectromelia, herpes simplex virus, cytomegalovirus, adenovirus and others. One of these compounds, hexadecyloxypropyl-CDV (HDP-CDV, CMX001) is in clinical development for prevention and treatment of poxvirus infection, vaccination complications, and for infections caused by cytomegalovirus, adenovirus, herpesviruses and other dsDNA viruses. This class of lipid analogs is potentially prone to undergo omega oxidation of the alkyl moiety which can lead to a short chain carboxylic acid lacking antiviral activity. To address this issue, we synthesized a series of alkoxyalkyl or alkyl glycerol esters of CDV and (S)-HPMPA having modifications in the structure of the alkyl residue. Antiviral activity was assessed in cells infected with vaccinia, cowpox or ectromelia viruses. Metabolic stability was determined in S9 membrane fractions from rat, guinea pig, monkey and human liver. All compounds had substantial antiviral activity in cells infected with vaccinia, cowpox or ectromelia. Metabolic stability was lowest in monkey liver S9 incubations where rapid disappearance of HDP-CDV and HDP-(S)-HPMPA was noted. Metabolic stability in monkey preparations increased substantially when a ω-1 methyl group (15-methyl-HDP-CDV) or a terminal cyclopropyl residue (14-cyclopropyl-tetradecyloxypropyl-CDV) was present in the alkyl chain. The most stable compound was 1-O-octadecyl-2-O-benzyl-sn-glycero-3-CDV (ODBG-CDV) which was not metabolized extensively by monkey liver S9. In rat, guinea pig or human liver S9 incubations, most of the modified antiviral compounds were considerably more stable.

SummaryEsterification of cidofovir (CDV), an antiviral nucleoside phosphonate, with alkyl or alkoxyalkyl groups increases antiviral activity by enhancing cell uptake and conversion to CDV diphosphate. Hexadecyloxypropyl-CDV (HDP-CDV) has been shown to be 40 to 100 times more active than CDV in vitro in cells infected with herpes group viruses, variola, cowpox, vaccinia or ectromelia viruses. Since the first phosphorylation of CDV may be rate limiting, we synthesized the hexadecyloxypropylphosphate (HDP-P-) and octadecyloxyethyl-phosphate (ODE-P-) conjugates of CDV and phosphonomethoxy-ethyl-adenine (PMEA, adefovir). We tested the CDV analogs in cells infected with human cytomegalovirus, herpes simplex virus, cowpox virus and vaccinia virus; the analogs of PMEA were tested in cells infected with the human immunodeficiency virus, type 1. In general, the alkoxyalkyl-phosphate conjugates of CDV were substantially more active than CDV. HDP-P-CDV and ODE-P-CDV were 4.6 to 40 times more active against HCMV and 7 to 30 times more active against cowpox and vaccinia in vitro. Although the compounds of this type were more cytotoxic than the unmodified bases, their selectivity for virally infected cells was generally greater than the parent nucleotides except that HDP-P-PMEA showed little or no selectivity in HIV-1 infected MT-2 cells. Although the new compounds with an interposed phosphate were generally less active that the corresponding alkoxyalkyl esters of CDV and PMEA, the present approach provides a possible alternative method for enhancing the antiviral activity of drugs of this class.