During heart development the second heart field (SHF) provides progenitor cells for most cardiomyocytes and expresses the homeodomain factor Nkx2-5. We now show that feedback repression of Bmp2/Smad1 signaling by Nkx2-5 critically regulates SHF proliferation and outflow tract (OFT) morphology. In the cardiac fields of Nkx2-5 mutants, genes controlling cardiac specification (including Bmp2) and maintenance of the progenitor state were upregulated, leading initially to progenitor overspecification, but subsequently to failed SHF proliferation and OFT truncation. In Smad1 mutants, SHF proliferation and deployment to the OFT were increased, while Smad1 deletion in Nkx2-5 mutants rescued SHF proliferation and OFT development. In Nkx2-5 hypomorphic mice, which recapitulate human congenital heart disease (CHD), OFT anomalies were also rescued by Smad1 deletion. Our findings demonstrate that Nkx2-5 orchestrates the transition between periods of cardiac induction, progenitor proliferation, and OFT morphogenesis via a Smad1-dependent negative feedback loop, which may be a frequent molecular target in CHD.

We have identified, isolated, and partially characterized doc-1, a novel cDNA sequence whose activity is consistent with a suppressor of hamster oral carcinogenesis. Doc-1 is an evolutionarily conserved gene exhibiting loss of heterozygosity and marked reduction in expression in malignant hamster oral keratinocytes. The full-length doc-1 cDNA encodes an 87 amino acid product that shows a significant homology to one of the seven novel genes induced in mouse fibroblasts by TNF-alpha. Transfection of the full-length doc-1 cDNA into malignant hamster oral keratinocytes alters the behavior of the recipients in terms of morphology, growth rate, and anchorage-independent growth, suggesting reversion of transformation phenotypes. We propose that doc-1 is a novel tumor suppressor gene in oral cancer development.

Regulated cyclin-dependent kinase (CDK) levels and activities are critical for the proper progression of the cell division cycle. p12 DOC-1 is a growth suppressor isolated from normal keratinocytes. We report that p12 DOC-1 associates with CDK2. More specifically, p12 DOC-1 associates with the monomeric nonphosphorylated form of CDK2 (p33CDK2). Ectopic expression of p12 DOC-1 resulted in decreased cellular CDK2 and reduced CDK2-associated kinase activities and was accompanied by a shift in the cell cycle positions of p12 DOC-1 transfectants (1 G 1 and 2 S). The p12 DOC-1 -mediated decrease of CDK2 was prevented if the p12 DOC-1 transfectants were grown in the presence of the proteosome inhibitor clasto-lactacystin ␤-lactone, suggesting that p12 DOC-1 may target CDK2 for proteolysis. A CDK2 binding mutant was created and was found to revert p12 DOC-1 -mediated, CDK2-associated cell cycle phenotypes. These data support p12 DOC-1 as a specific CDK2-associated protein that negatively regulates CDK2 activities by sequestering the monomeric pool of CDK2 and/or targets CDK2 for proteolysis, reducing the active pool of CDK2. Cell cycle inhibitors of the p16INK4a and p21families exert their effects by negatively regulating cyclin and cyclin-dependent kinase (CDK) complex formation and kinase activities (10,14). While the p16 INK4a family is specific for CDK4 and CDK6, and the p21 WAF1/CIP1/CAP20 family of CDK inhibitors is universal for CDKs, there is no known specific inhibitor for CDK2. CDK2, when complexed with cyclins E and A, is implicated in G 1 /S transition, DNA replication, and progression through the DNA synthesis phase (6, 7, 9). p12 DOC-1 is a growth suppressor identified and isolated from normal keratinocytes (12). It is a highly conserved cellular gene. Our laboratory (12, 13) and others (4, 5) have cloned p12 DOC-1 cDNA from human, mouse, and hamster. The fulllength human and mouse p12 DOC-1 cDNAs are 1.6 kb and 1.2 kb, respectively. Human p12 DOC-1 has one additional amino acid at residue 19, which corresponds to an alanine, and differs from the mouse and hamster p12 DOC-1 at only two other amino acid residues (Ala 3 Thr at residue 8 and Gly 3 Ser at residue 100). Human and rodent p12 DOC-1 polypeptides have 97% identity, and the mouse and hamster p12 DOC-1 protein sequences are identical. Human p12 DOC-1 is a 115-amino-acid peptide with a molecular mass of 12.4 kDa (pI, 9.62).Ectopic expression of p12 DOC-1 in keratinocytes is associated with increased doubling time, suggestive of a growth suppressor function (11). These observations prompted us to examine if p12 DOC-1 interacts with regulatory proteins in the cell division cycle. We report that p12 DOC-1 associates with CDK2. Data are presented to support the role of p12 DOC-1 as a specifically CDK2-associated protein, which, when overexpressed, negatively regulates CDK2-associated kinase activities and cell cycle phenotypes. MATERIALS AND METHODSCell culture and transfections. Transfection of human 293 cells was performed using Lipofectamine Plus (Life...

SummaryTransforming growth factor a (TGF-a) is a pleuripotential cytokine with diverse biological effects, including the ability to influence the proliferation of normal cells or neoplastic epithelial cells. Eosinophils are a subset of granulocytes that normally enter the peripheral tissues, particularly those beneath gastrointestinal, respiratory, and urogenital epithelium, where they reside in close proximity to the epithelial elements . In this study, we demonstrate that the great majority of eosinophils infiltrating the interstitial tissues adjacent to two colonic adenocarcinomas and two oral squamous cell carcinomas labeled specifically by in situ hybridization with a 35S-riboprobe for human TGF-a (hTGF-a) . No other identifiable leukocytes in these lesions contained detectable hTGF-a mRNA. We also examined leukocytes purified from a patient with the idiopathic hypereosinophilic syndrome. 80% of these eosinophils, but none of the patient's neutrophils or mononuclear cells, were positive for hTGF-a mRNA by in situ hybridization, and 55% of these eosinophils were positive by immunohistochemistry with a monoclonal antibody directed against the COOH terminus of the mature hTGF-a peptide. Finally, the identification of the purified eosinophil-associated transcript as hTGF-ca was confirmed by polymerase chain reaction product restriction enzyme analysis followed by Southern blot hybridization . In contrast to eosinophils from the patient with hypereosinophilic syndrome, the peripheral blood eosinophils from only two of seven normal donors had detectable TGF-a mRNA and none ofthese eosinophils contained immunohistochemically detectable TGF-a product. Taken together, these findings establish that human eosinophils can express TGF-a, but suggest that the expression of TGF-a by eosinophils may be under microenvironmental regulation. Demonstration of TGF-a production by tissueinfiltrating eosinophils and the eosinophils in the hypereosinophilic syndrome identifies a novel mechanism by which eosinophils might contribute to physiological, immunological, and pathological responses .

doc-1 is a putative tumor suppressor gene isolated and identified from the hamster oral cancer model. Here, we report the molecular cloning and the functional characterization of the human ortholog of the hamster doc-1 gene. Human doc-1 cDNA is 1.6 kilobase pairs in length and encodes for a 115-amino acid polypeptide (12.4 kDa, pI 9.53). Sequence analysis showed 98% identity between human and hamster doc-1 protein sequences. DOC-1 is expressed in all normal human tissues examined. In oral keratinocytes, expression of DOC-1 is restricted to normal oral keratinocytes. By immunostaining of normal human mucosa, DOC-1 is detected in both the cytoplasm and nuclei of basal oral keratinocytes; while in suprabasilar cells, it is primarily found in the nuclei. Human oral cancers in vivo did not exhibit immunostaining for DOC-1. Like murine DOC-1, human DOC-1 associates with DNA polymerase ␣/primase and mediates the phosphorylation of the large p180 catalytic subunit, suggesting it may be a potential regulator of DNA replication in the S phase of the cell cycle. Using a human doc-1 cosmid as a probe, human doc-1 is mapped to chromosome 12q24. We identified four exons in the entire human doc-1 gene and determined the intron-exon boundaries. By polymerase chain reaction and direct sequencing, we examined premalignant oral lesion and oral cancer cell lines and found no intragenic mutations. Squamous cell carcinoma (SCC)1 of the oral cavity is newly diagnosed in 38,000 Americans each year and in 350,000 people worldwide (1, 2). Approximately half of the patients afflicted die within 5 years of diagnosis, while surviving patients may be left with severe cosmetic and/or functional compromise (1-3). Survival curves of oral cancer patients have plateaued over the past 2 decades and remained among the worst of all cancer sites.The hamster oral cancer model is an excellent model to study the molecular event during oral carcinogenesis (4 -7). doc-1 is a putative tumor suppressor gene identified and isolated from the carcinogen-induced hamster oral cancer model (8). DOC-1 is predicted to be a 114-amino acid peptide with a molecular mass of 12.4 kDa. Transfection of doc-1 into malignant oral keratinocytes led to the reversion of transformation phenotypes including anchorage independence, doubling time, and morphology. The genetic sequence of doc-1 matched to a tumor necrosis factor-␣-induced early-response murine transcript, TU-166 (9), suggesting that doc-1 may be a downstream event in the tumor necrosis factor-␣ signaling pathway. We have recently cloned the full-length mouse doc-1 cDNA (GenBank TM number AF011644); its DNA sequence in the open reading frame is 94% identical to that of the hamster. The predicted amino acid peptides encoded by the mouse and hamster doc-1 open reading frames are identical. Each open reading frame encodes for a 114-amino acid peptide that has a predicted molecular mass of 12.4 kDa and a pI of 9.53.The highly conserved nature of the rodent doc-1 genes prompted us to clone and examine the role of doc-1 in...