PTEN is among the most frequently inactivated tumour suppressor genes in sporadic cancer. PTEN has dual protein and lipid phosphatase activity, and its tumour suppressor activity is dependent on its lipid phosphatase activity, which negatively regulates the PI3K-AKT-mTOR pathway. Germline mutations in PTEN have been described in a variety of rare syndromes that are collectively known as the PTEN hamartoma tumour syndromes (PHTS). Cowden syndrome is the best-described syndrome within PHTS, with approximately 80% of patients having germline PTEN mutations. Patients with Cowden syndrome have an increased incidence of cancers of the breast, thyroid and endometrium, which correspond to sporadic tumour types that commonly exhibit somatic PTEN inactivation. Pten deletion in mice leads to Cowden syndrome-like phenotypes, and tissue-specific Pten deletion has provided clues to the role of PTEN mutation and loss in specific tumour types. Studying PTEN in the continuum of rare syndromes, common cancers and mouse models provides insight into the role of PTEN in tumorigenesis and will inform targeted drug development.

G 1 ͞S and G 2 ͞M cell cycle checkpoints maintain genomic stability in eukaryotes in response to genotoxic stress. We report here both genetic and functional evidence of a Gadd45-mediated G 2 ͞M checkpoint in human and murine cells. Increased expression of Gadd45 via microinjection of an expression vector into primary human fibroblasts arrests the cells at the G 2 ͞M boundary with a phenotype of MPM2 immunopositivity, 4n DNA content and, in 15% of the cells, centrosome separation. The Gadd45-mediated G 2 ͞M arrest depends on wild-type p53, because no arrest was observed either in p53-null Li-Fraumeni fibroblasts or in normal fibroblasts coexpressed with p53 mutants. Increased expression of cyclin B1 and Cdc25C inhibited the Gadd45-mediated G 2 ͞M arrest in human fibroblasts, indicating that the mechanism of Gadd45-mediated G 2 ͞M checkpoint is at least in part through modulation of the activity of the G 2 -specific kinase, cyclin B1͞p34 cdc2 . Genetic and physiological evidence of a Gadd45-mediated G 2 ͞M checkpoint was obtained by using GADD45-deficient human or murine cells. Human cells with endogenous Gadd45 expression reduced by antisense GADD45 expression have an impaired G 2 ͞M checkpoint after exposure to either ultraviolet radiation or methyl methanesulfonate but are still able to undergo G 2 arrest after ionizing radiation. Lymphocytes from gadd45-knockout mice (gadd45 ؊͞؊) also retained a G 2 ͞M checkpoint initiated by ionizing radiation and failed to arrest at G 2 ͞M after exposure to ultraviolet radiation. Therefore, the mammalian genome is protected by a multiplicity of G 2 ͞M checkpoints in response to specific types of DNA damage.Mammalian cells have evolved an intricate defense network to maintain genomic integrity by preventing the fixation of permanent damage from endogenous and exogenous mutagens. Cellcycle checkpoints, a major genomic surveillance mechanism, exist at the G 1 ͞S and G 2 ͞M transitions that are regulated in response to DNA damage (1). Defects in these steps may result in a mutator phenotype that is associated with tumorigenesis.Tumor suppressor gene product p53 is implicated to be one of the essential components of cell-cycle checkpoints (2-5). p53 is a transcription factor that up-regulates a number of important cell cycle-modulating genes, including p21 WAF1͞CIP1͞SDI1

More than 20 different cDNA clones encoding DNA-damage-inducible transcripts in rodent cells have recently been isolated by hybridization subtraction (A. J. Fornace, Jr., I. Alamo, Jr., and M. C. Hollander, Proc. Natl. Acad. Sci. USA 85:8800-8804, 1988). In most cells, one effect of DNA damage is the transient inhibition of DNA synthesis and cell growth. We now show that five of our clones encode transcripts that are increased by other growth cessation signals: growth arrest by serum reduction, medium depletion, contact inhibition, or a 24-h exposure to hydroxyurea. The genes coding for these transcripts have been designated gadd (growth arrest and DNA damage inducible). Two of the gadd cDNA clones were found to hybridize at high stringency to transcripts from human cells that were induced after growth cessation signals or treatment with DNA-damaging agents, which indicates that these responses have been conserved during mammalian evolution. In contrast to results with growth-arrested cells that still had the capacity to grow after removal of the growth arrest conditions, no induction occurred in HL60 cells when growth arrest was produced by terminal differentiation, indicating that only certain kinds of growth cessation signals induce these genes. All of our experiments suggest that the gadd genes are coordinately regulated: the kinetics of induction for all five transcripts were similar; in addition, overexpression of gadd genes was found in homozygous deletion c14CoS/c14CoS mice that are missing a small portion of chromosome 7, suggesting that a trans-acting factor encoded by a gene in this deleted portion is a negative effector of the gadd genes. The gadd genes may represent part of a novel regulatory pathway involved in the negative control of mammalian cell growth.

Hybridization subtraction at low ratios of RNA to cDNA was used to enrich for the cDNA of transcripts increased in Chinese hamster cells after UV irradiation. Forty-nine different cDNA clones were isolated. Most coded for nonabundant transcripts rapidly induced 2-to 10-fold after UV irradiation. Only 2 of the 20 cDNA clones sequenced matched known sequences (metallothionein I and II). The predicted amino acid sequence of one cDNA had two localized areas of homology with the rat helix-destabilizing protein. These areas of homology were at the two DNA-binding sites of this nucleic acid single-strand-binding protein. The induced transcripts were separated into two general classes. Class I transcripts were induced by UV radiation and not by the alkylating agent methyl methanesulfonate. Class II transcripts were induced by UV radiation and by methyl methanesulfonate. Many class II transcripts were induced also by H202 and various alkylating agents but not by heat shock, phorbol 12-tetradecanoate 13-acetate, or DNA-damaging agents which do not produce high levels of base damage. Since many of the cDNA clones coded for transcripts which were induced rapidly and only by certain types of DNA-damaging agents, their induction is likely a specific response to such damage rather than a general response to cell inJury.In Escherichia coli, DNA damage caused by UV radiation or chemical agents induces approximately 20 genes of the SOS regulon; many of these genes code for proteins involved in DNA repair, mutagenesis, and recombination (1). Damage by alkylating agents or oxidative stress induces other wellcharacterized regulons in bacteria (1). In yeast, induction by UV radiation of the RAD2 excision-repair gene is similar to induction of the SOS response in bacteria: the transcript is of low abundance, and its induction is rapid and only 2-to 10-fold (2). The RAD2 gene is likely only one of many yeast DNA-damage-inducible (DDI) genes; Ruby and Szostak (3) have estimated there may be up to 80 such genes. In mammalian cells only a few DDI genes (4-6) have been identified; the cDNA clones of these genes were isolated by differential screening, which is effective for detecting the cDNA of only abundant transcripts [abundance > 0.1% of poly(A)+ RNA] (ref. 7, p. 226). These mammalian genes differed also from the RAD2 and SOS genes in that the former were induced slowly.To determine if low-abundance DDI transcripts occur in mammalian cells, an approach is required to enrich for, and isolate, the cDNA of such transcripts. Standard hybridization subtraction, which employs a high ratio of RNA to cDNA, is an effective approach to enrich for the cDNA of lowabundance transcripts that are expressed at much higher levels in one cell type than in another (ref. 7, p. 228). We have previously shown that hybridization subtraction using low ratios of RNA to cDNA enriches for the cDNA of transcripts increased only a fewfold over control levels (8): when cDNA synthesized from the RNA of heat-shocked cells was hybridized with an equal amount o...

Gadd45a-null mice generated by gene targeting exhibited several of the phenotypes characteristic of p53-deficient mice, including genomic instability, increased radiation carcinogenesis and a low frequency of exencephaly. Genomic instability was exemplified by aneuploidy, chromosome aberrations, gene amplification and centrosome amplification, and was accompanied by abnormalities in mitosis, cytokinesis and growth control. Unequal segregation of chromosomes due to multiple spindle poles during mitosis occurred in several Gadd45a -/- cell lineages and may contribute to the aneuploidy. Our results indicate that Gadd45a is one component of the p53 pathway that contributes to the maintenance of genomic stability.

A remarkable overlap was observed between the gadd genes, a group of often coordinately expressed genes that are induced by genotoxic stress and certain other growth arrest signals, and the MyD genes, a set of myeloid differentiation primary response genes. The MyD116 gene was found to be the murine homolog of the hamster gadd34 gene, whereas MyD118 and gadd45 were found to represent two separate but closely related genes. Furthermore, gadd34/MyD116, gadd45, MyD118, and gadd153 encode acidic proteins with very similar and unusual charge characteristics; both this property and a similar pattern of induction are shared with mdm2, which, like gadd45, has been shown previously to be regulated by the tumor suppressor p53. Expression analysis revealed that they are distinguished from other growth arrest genes in that they are DNA damage inducible and suggests a role for these genes in growth arrest and apoptosis either coupled with or uncoupled from terminal differentiation. Evidence is also presented for coordinate induction in vivo by stress. The use of a short-term transfection assay, in which expression vectors for one or a combination of these gadd/MyD genes were transfected with a selectable marker into several different human tumor cell lines, provided direct evidence for the growth-inhibitory functions of the products of these genes and their ability to synergistically suppress growth. Taken together, these observations indicate that these genes define a novel class of mammalian genes encoding acidic proteins involved in the control of cellular growth.

Purpose: The development of new cancer drugs is slow and costly. HIV protease inhibitors are Food and Drug Administration approved for HIV patients. Because these drugs cause toxicities that can be associated with inhibition of Akt, an emerging target in cancer, we assessed the potential of HIV protease inhibitors as anticancer agents. Experimental Design: HIV protease inhibitors were screened in vitro using assays that measure cellular proliferation, apoptotic and nonapoptotic cell death, endoplasmic reticulum (ER) stress, autophagy, and activation of Akt. Nelfinavir was tested in non–small cell lung carcinoma (NSCLC) xenografts with biomarker assessment. Results: Three of six HIV protease inhibitors, nelfinavir, ritonavir, and saquinavir, inhibited proliferation of NSCLC cells, as well as every cell line in the NCI60 cell line panel. Nelfinavir was most potent with a mean 50% growth inhibition of 5.2 μmol/L, a concentration achievable in HIV patients. Nelfinavir caused two types of cell death, caspase-dependent apoptosis and caspase-independent death that was characterized by induction of ER stress and autophagy. Autophagy was protective because an inhibitor of autophagy increased nelfinavir-induced death. Akt was variably inhibited by HIV protease inhibitors, but nelfinavir caused the greatest inhibition of endogenous and growth factor–induced Akt activation. Nelfinavir decreased the viability of a panel of drug-resistant breast cancer cell lines and inhibited the growth of NSCLC xenografts that was associated with induction of ER stress, autophagy, and apoptosis. Conclusions: Nelfinavir is a lead HIV protease inhibitor with pleiotropic effects in cancer cells. Given its wide spectrum of activity, oral availability, and familiarity of administration, nelfinavir is a Food and Drug Administration–approved drug that could be repositioned as a cancer therapeutic.

Human cells lacking functional p53 exhibit a partial deficiency in nucleotide excision repair (NER), the pathway for repair of UV-induced DNA damage. The global genomic repair (GGR) subpathway of NER, but not transcription-coupled repair (TCR), is mainly affected by p53 loss or inactivation. We have utilized mouse embryo fibroblasts (MEFs) lacking p53 genes or downstream effector genes of the p53 pathway, gadd45 (Gadd45a) or p21 (Cdkn1a), as well as MEFs lacking both gadd45 and p21 genes to address the potential contribution of these downstream effectors to p53-associated DNA repair. Loss of p53 or gadd45 had a pronounced effect on GGR, while p21 loss had only a marginal effect, determined by measurements of repair synthesis (unscheduled DNA synthesis), by immunoassays to detect removal of UV photoproducts from genomic DNA, and by assays determining strand-specific removal of CPDs from the mouse dhfr gene. Taken together, the evidence suggests a role for Gadd45, but relatively little role for p21, in DNA repair responses to UV radiation. Recent evidence suggests that Gadd45 binds to UV-damaged chromatin and may affect lesion accessibility. MEFs lacking p53 or gadd45 genes exhibited decreased colony-forming ability after UV radiation and cisplatin compared to wildtype MEFs, indicating their sensitivity to DNA damage. We provide evidence that Gadd45 affects chromatin remodelling of templates concurrent with DNA repair, thus indicating that Gadd45 may participate in the coupling between chromatin assembly and DNA repair.