The basic leucine zipper transcription factor CCAAT͞enhancer binding protein-␤ (C͞EBP␤) is expressed in many cell types, including keratinocytes. C͞EBP␤ activity can be increased by phosphorylation through pathways stimulated by oncogenic Ras, although the biological implications of Ras-C͞EBP␤ signaling are not currently understood. We report here that C͞EBP␤-nullizygous mice are completely refractory to skin tumor development induced by a variety of carcinogens and carcinogenesis protocols, including 7,12-dimethylbenz-[a]anthracene-initiation͞12-O-tetradecanoylphorbol 13-acetate promotion, that produce tumors containing oncogenic Ras mutations. No significant differences in TPA-induced epidermal keratinocyte proliferation were observed in C͞EBP␤-null versus wild-type mice. However, apoptosis was significantly elevated (17-fold) in the epidermal keratinocytes of 7,12-dimethylbenz[a]anthracene-treated C͞EBP␤-null mice compared with wild-type mice. In v-Ha-ras transgenic mice, C͞EBP␤ deficiency also led to greatly reduced skin tumor multiplicity and size, providing additional evidence for a tumorigenesis pathway linking Ras and C͞EBP␤. Oncogenic Ras potently stimulated C͞EBP␤ to activate a C͞EBP-responsive promoter-reporter in keratinocytes and mutating an ERK1͞2 phosphorylation site (T188) in C͞EBP␤ abolished this Ras effect. Finally, we observed that C͞EBP␤ participates in oncogenic Ras-induced transformation of NIH 3T3 cells. These findings indicate that C͞EBP␤ has a critical role in Ras-mediated tumorigenesis and cell survival and implicate C͞EBP␤ as a target for tumor inhibition.T he Ras family of GTP binding proteins function as intracellular mediators of extracellular signals to regulate cell proliferation, apoptosis, survival, senescence, and differentiation (1-5). Ras protooncogenes are frequently mutated in tumors, and Ϸ25% of human cancers contain transforming mutations in ras. Therefore, understanding oncogenic Ras-signaling pathways is critical for elucidating the mechanisms that underlie cellular transformation and for designing effective therapeutic strategies to prevent the development or block the growth of many classes of tumors. Ras has numerous effectors, and its pathways are multifaceted (3, 6, 7). Ras activation by growth factors or oncogenic mutations elicits activation of several transcription factors, which in turn regulate the expression of genes that control the cellular responses to Ras signaling, including oncogenesis. The transcription factors Ets, c-jun, c-myc, and NF-B are known to have roles in oncogenic ras-induced cellular transformation (8-11).The basic leucine zipper (bZIP) transcription factor CCAAT͞ enhancer binding protein-␤ (C͞EBP␤, also known as NF-IL6, IL-6DBP, LAP, CRP2, and NF-M) is expressed in a variety of cell types (12, 13) including keratinocytes (14,15), where it plays a role in squamous differentiation (16). C͞EBP␤ is also involved in regulating differentiation of specific mesenchymal, epithelial, and hematopoietic cell types (17-21). C͞EBP␤ activity can be activ...

The epidermis is a stratified squamous epithelium composed primarily of keratinocytes that become postmitotic and undergo sequential changes in gene expression during terminal differentiation. The expression of the transcription factor CCAAT/enhancer binding protein ␤ (C/EBP␤) within mouse epidermis and primary keratinocytes has recently been described; however, the function of C/EBP␤ within the epidermal keratinocyte is unknown. We report here that transient transfection of mouse primary keratinocytes with a C/EBPresponsive promoter-reporter construct resulted in a sevenfold increase in luciferase activity when keratinocytes were switched to culture conditions that induce growth arrest and differentiation. Forced expression of C/EBP␤ in BALB/MK2 keratinocytes inhibited growth, induced morphological changes consistent with a more differentiated phenotype, and upregulated two early markers of differentiation, keratin 1 (K1) and keratin 10 (K10) but had a minimal effect on the expression of late-stage markers, loricrin and involucrin. Analysis of the epidermis of C/EBP␤-deficient mice revealed a mild epidermal hyperplasia and decreased expression of K1 and K10 but not of involucrin and loricrin. C/EBP␤-deficient primary keratinocytes were partially resistant to calcium-induced growth arrest. Analysis of terminally differentiated spontaneously detached keratinocytes or those induced to differentiate by suspension culture revealed that C/EBP␤-deficient keratinocytes displayed striking decreases in K1 and K10, while expression of later-stage markers was only minimally altered. Our results demonstrate that C/EBP␤ plays an important role in the early events of stratified squamous differentiation in keratinocytes involving growth arrest and K1 and K10 expression.

Black tea extracts (hot aqueous, polyphenols and theaflavins) and green tea extracts (hot aqueous, polyphenols, epicatechin, epicatechin gallate, epigallocatechin and epigallocatechin gallate) were tested in nine standardized cell culture assays for comparative cancer chemopreventive properties. Most black and green tea extracts strongly inhibited neoplastic transformation in mouse mammary organ cultures, rat tracheal epithelial cells and human lung tumor epithelial cells. Nearly all tea fractions strongly inhibited benzo[a]pyrene adduct formation with human DNA. Induction of phase II enzymes, glutathione-S-transferase and quinone reductase, were enhanced by nearly all tea fractions, while glutathione was induced by only a few fractions. Ornithine decarboxylase activity was inhibited by nearly all the green tea fractions, but none of the black tea fractions. 12-O-tetradecanoylphorbol-13-acetate-induced free radicals were inhibited by most tea fractions. These results provide strong evidence of both anti-mutagenic, anti-proliferative and anti-neoplastic activities for both black and green tea extracts. Such anticancer mechanisms may well be responsible for the cancer preventive efficacies seen in both experimental and human studies.

CCAAT/enhancer binding protein ␤ (C/EBP␤) is a widely expressed transcription factor whose activity is regulated by oncogenic Ha-Ras V12 signaling. C/EBP␤ is essential for the development of mouse skin tumors containing Ras mutations and can cooperate with Ras V12 to transform NIH 3T3 cells. Here we have investigated Ras-induced phosphorylation of C/EBP␤ in fibroblasts and report a novel proline-directed phosphoacceptor site at Ser64 within the transactivation domain. Ser64 phosphorylation was induced by activated Ras and Raf but was not blocked by chemical inhibitors of MEK1/2, phosphatidylinositol 3-kinase, JNK, or p38 mitogenactivated protein kinases. Ser64 was efficiently phosphorylated in vitro by the cyclin-dependent kinases Cdk2 and Cdc2. Thr189, previously identified as an ERK1/2 phosphorylation site that regulates C/EBP␤ activity, was also a substrate for Cdk phosphorylation. Ser64 and Thr189 phosphorylation was low in serum-starved (G 0 ) cells but was strongly increased in mid-G 1 cells and in cells arrested in S or M phase. In addition, phosphorylation on both sites was blocked by treating cells with the Cdk inhibitor roscovitine. In contrast to wild-type C/EBP␤, which enhances transformation of NIH 3T3 cells, mutants bearing alanine substitutions at Ser64 and/or Thr189 inhibited Ras V12 -induced focus formation. Our findings support a role for C/EBP␤ as a nuclear effector of Ras signaling and transformation, and they indicate that cell cycle-dependent phosphorylation of C/EBP␤ on Ser64 and Thr189 is required to promote Ras-induced transformation of NIH 3T3 cells.

We studied a series of 64 patients with sensory +/- motor peripheral neuropathies by comparing clinical and physiologic features to serum antibody reactivity against compounds containing sulfated carbohydrate moieties. We determined antibody reactivity by an enzyme-linked immunosorbent assay (ELISA) using purified glycolipids and glycoproteins as antigens, and we used high-performance thin-layer chromatography and Western blotting to test the specificity of results. Twelve patients with high titers of IgM antibodies directed against the myelin-associated glycoprotein (MAG) had sensory-motor polyneuropathies with physiologic evidence of demyelination. IgM antibody reactivity to MAG was associated with an IgM serum M protein in five patients. Eight other patients, most with sensory greater than motor polyneuropathies, had high titers of antibody reactivity to sulfatide but not of IgM to MAG. Two had an associated IgM paraprotein. None of the patients with selective serum antisulfatide activity had predominantly demyelinating features on physiologic testing. We conclude that (1) high ELISA titers of antibodies to MAG may be more common than previously suspected in patients with chronic demyelinating sensory-motor neuropathies, and (2) the presence of high titers of antisulfatide antibodies in serum may provide clues to the pathogenesis of otherwise idiopathic, axonal, predominantly sensory neuropathies.

Autoimmune lymphoproliferative syndrome (ALPS) is characterized by lymphadenopathy, elevated numbers of T cells with alphabeta-T cell receptors but neither CD4 nor CD8 co-receptors, and impaired lymphocyte apoptosis in vitro. Defects in the Fas receptor are the most common cause of ALPS (ALPS Ia), but in rare cases other apoptosis proteins have been implicated, including caspase-10 (ALPS II). We investigated the role of variants of caspase-10 in ALPS. Of 32 unrelated probands with ALPS who did not have Fas defects, two were heterozygous for the caspase-10 missense mutation I406L. Like the previously reported ALPS II-associated mutation L285F, I406L impaired apoptosis when transfected alone and dominantly inhibited apoptosis mediated by wild type caspase-10 in a co-transfection assay. Other variants in caspase-10, V410I and Y446C, were found in 3.4 and 1.6% of chromosomes in Caucasians, and in 0.5 and <0.5% of African Americans, respectively. In contrast to L285F and I406L, these variants had no dominant negative effect in co-transfection assays into the H9 lymphocytic cell line. We found healthy individuals homozygous for V410I, challenging the earlier suggestion that homozygosity for V410I alone causes ALPS. Moreover, an association analysis suggested protection from severe disease by caspase-10 V410I in 63 families with ALPS Ia due to dominant Fas mutations (P<0.05). Thus, different genetic variations in caspase-10 can produce contrasting phenotypic effects.

The CCAAT/enhancer binding protein b (C/EBPb) is implicated in the regulation of many different molecular and physiological processes. Mice with a germline deletion of C/EBPb (C/EBPb À/À ) display phenotypes in a multitude of cell types and organ systems, including skin where C/EBPb À/À mice exhibit increased apoptosis in epidermal keratinocytes in response to carcinogen treatment and are completely resistant to carcinogen-induced skin tumorigenesis. To determine the contribution of systemic versus cell autonomous functions of C/EBPb to specific phenotypes, mice with a conditional 'floxed' C/EBPb null allele were generated. Epidermal-specific deletion of C/EBPb was achieved by Cre recombinase expression from a keratin 5 (K5) promoter. Similar to C/EBPb À/À mice, K5-Cre;C/ EBPb fl/fl mice were completely refractory to 7,12 dimethylbenz[a]anthracene (DMBA)-induced skin tumorigenesis and these mice displayed increased DMBA-induced apoptosis in epidermal keratinocytes compared to wild-type mice. In contrast, mice lacking the related gene, C/EBPd, were not resistant to DMBA-induced skin tumorigenesis, indicating a unique role of C/EBPb in skin tumor development. Our findings demonstrate that C/EBPb exerts an essential, keratinocyte-intrinsic role in cell survival in response to carcinogen treatment and the elimination of C/EBPb in keratinocytes is sufficient to confer complete resistance of the skin to chemical carcinogenesis.

CCAAT/enhancer binding protein A (C/EBPA) is a basic leucine zipper transcription factor that inhibits cell cycle progression and regulates differentiation in various cell types. C/EBPA is inactivated by mutation in acute myeloid leukemia (AML) and is considered a human tumor suppressor in AML. Although C/EBPA mutations have not been observed in malignancies other than AML, greatly diminished expression of C/EBPA occurs in numerous human epithelial cancers including lung, liver, endometrial, skin, and breast, suggesting a possible tumor suppressor function. However, direct evidence for C/EBPA as an epithelial tumor suppressor is lacking due to the absence of C/EBPA mutations in epithelial tumors and the lethal effect of C/EBPA deletion in mouse model systems. To examine the function of C/EBPA in epithelial tumor development, an epidermal-specific C/EBPA knockout mouse was generated. The epidermal-specific C/EBPA knockout mice survived and displayed no detectable abnormalities in epidermal keratinocyte proliferation, differentiation, or apoptosis, showing that C/EBPA is dispensable for normal epidermal homeostasis. In spite of this, the epidermal-specific C/EBPA knockout mice were highly susceptible to skin tumor development involving oncogenic Ras. These mice displayed decreased tumor latency and striking increases in tumor incidence, multiplicity, growth rate, and the rate of malignant progression. Mice hemizygous for C/ EBPA displayed an intermediate-enhanced tumor phenotype. Our results suggest that decreased expression of C/EBPA contributes to deregulation of tumor cell proliferation. C/ EBPA had been proposed to block cell cycle progression through inhibition of E2F activity. We observed that C/EBPA blocked Ras-induced and epidermal growth factor-induced E2F activity in keratinocytes and also blocked Ras-induced cell transformation and cell cycle progression. Our study shows that C/EBPA is dispensable for epidermal homeostasis and provides genetic evidence that C/EBPA is a suppressor of epithelial tumorigenesis. [Cancer Res 2007;67(14):6768-76]