Cyclin-dependent Kinase Inhibitor CDKN1A Research Articles

Miz1 Is a Critical Repressor of cdkn1a during Skin Tumorigenesis

The transcription factor Miz1 forms repressive DNA-binding complexes with the Myc, Gfi-1 and Bcl-6 oncoproteins. Known target genes of these complexes encode the cyclin-dependent kinase inhibitors (CKIs) cdkn2b (p15Ink4), cdkn1a (p21Cip1), and cdkn1c (p57Kip2). Whether Miz1-mediated repression is important for control of cell proliferation in vivo and for tumor formation is unknown. Here we show that deletion of the Miz1 POZ domain, which is critical for Miz1 function, restrains the development of skin tumors in a model of chemically-induced, Ras-dependent tumorigenesis. While the stem cell compartment appears unaffected, interfollicular keratinocytes lacking functional Miz1 exhibit a reduced proliferation and an accelerated differentiation of the epidermis in response to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Tumorigenesis, proliferation and normal differentiation are restored in animals lacking cdkn1a, but not in those lacking cdkn2b. Our data demonstrate that Miz1-mediated attenuation of cell cycle arrest pathways via repression of cdkn1a has a critical role during tumorigenesis in the skin.

Ligand-dependent Corepressor (LCoR) Recruitment by Krüppel-like Factor 6 (KLF6) Regulates Expression of the Cyclin-dependent Kinase Inhibitor CDKN1A Gene

The widely expressed transcriptional coregulator, ligand-dependent corepressor (LCoR), initially characterized as a regulator of nuclear receptor-mediated transactivation, functions through recruitment of C-terminal binding proteins (CtBPs) and histone deacetylases (HDACs) to its N-terminal and central domains, respectively. We performed a yeast two-hybrid screen for novel cofactors, and identified an interaction between the C-terminal domain of LCoR and the transcription factor Krüppel-like factor 6 (KLF6), a putative tumor suppressor in prostate cancer. Subsequent experiments revealed LCoR regulation of several KLF6 target genes notably p21(WAF1/CIP1) (CDKN1A) and to a lesser extent E-cadherin (CDH1), indicating that LCoR regulates gene transcription through multiple classes of transcription factors. In multiple cancer cells, LCoR and KLF6 bind together on the promoters of the genes encoding CDKN1A and CDH1. LCoR contributes to KLF6-mediated transcriptional repression in a promoter- and cell type-dependent manner. Its inhibition of reporter constructs driven by the CDKN1A and CDH1 promoters in PC-3 prostate carcinoma cells is sensitive to treatment with the HDAC inhibitor trichostatin A. Additionally, the LCoR cofactor CtBP1 bound the same promoters and augmented the LCoR-dependent repression in PC-3 cells. Consistent with their inferred roles in transcriptional repression, siRNA-mediated knockdown of KLF6, LCoR, or CtBP1 in PC-3 cells induced expression of CDKN1A and CDH1 and additional KLF6 target genes. We propose a novel model of LCoR function in which promoter-bound KLF6 inhibits transcription of the CDKN1A gene and other genes as well by tethering a transcriptional corepressor complex containing LCoR, with specific contributions by CtBP1 and HDACs.

Stress-Regulated Transcription Factor ATF4 Promotes Neoplastic Transformation by Suppressing Expression of the INK4a/ARF Cell Senescence Factors

Many cancers overexpress ATF4, a stress-induced transcription factor that promotes cell survival under hypoxic conditions and other stresses of the tumor microenvironment, but the potential contributions of ATF4 to oncogenesis itself have been little explored. Here, we report that ATF4 promotes oncogene-induced neoplastic transformation by suppressing the expression of cellular senescence-associated genes. Strikingly, primary embryo fibroblasts from ATF4-deficient mice were resistant to transformation by coexpression of H-ras(V12) and SV40 large T antigen. In wild-type cells these oncogenes induced expression of the murine Atf4 gene along with the cyclin-dependent kinase inhibitor Cdkn2a, which encodes the cell senescence-associated proteins p16INK4 and p19ARF. Elevated levels of ATF4 were sufficient to suppress expression of these proteins and drive oncogenic transformation. Conversely, genetic ablation of ATF4 led to constitutive expression of p16INK4a and p19ARF, triggering cellular senescence. Our findings define a central function for ATF4 in promoting oncogenic transformation by suppressing a central pathway of cellular senescence.

Genes and Abdominal Aortic Aneurysm

Abdominal aortic aneurysm (AAA) is a multifactorial disease with a strong genetic component. Since the first candidate gene studies were published 20 years ago, approximately 100 genetic association studies using single nucleotide polymorphisms (SNPs) in biologically relevant genes have been reported on AAA. These studies investigated SNPs in genes of the extracellular matrix, the cardiovascular system, the immune system, and signaling pathways. Very few studies were large enough to draw firm conclusions and very few results could be replicated in another sample set. The more recent unbiased approaches are family-based DNA linkage studies and genome-wide genetic association studies, which have the potential of identifying the genetic basis for AAA, only when appropriately powered and well-characterized large AAA cohorts are used. SNPs associated with AAA have already been identified in these large multicenter studies. One significant association was of a variant in a gene called contactin-3, which is located on chromosome 3p12.3. However, two follow-up studies could not replicate this association. Two other SNPs, which are located on chromosome 9p21 and 9q33, were replicated in other samples. The two genes with the strongest supporting evidence of contribution to the genetic risk for AAA are the CDKN2BAS gene, also known as ANRIL, which encodes an antisense ribonucleic acid that regulates expression of the cyclin-dependent kinase inhibitors CDKN2A and CDKN2B, and DAB2IP, which encodes an inhibitor of cell growth and survival. Functional studies are now needed to establish the mechanisms by which these genes contribute toward AAA pathogenesis.

Caffeic Acid Phenetyl Ester, a Brazilian-Green-Propolis Derivative, Induces apoptosis in AML Cells, Promotes up Regulation of G-Protein Signaling and Hyper Secretion of IL-8

AbstractAbstract 3274Propolis is a generic name for an adhesive resin collected, processed and used by bees to plug gaps, smooth internal walls and protect the entrance of the hive from intruders. Chemically is a complex blend of resin and fragments of plant tissues, volatile substances and wax. It contains over 300 constituents including benzoic acids, flavonoids and cinnamic acid derivatives. The Brazilian green propolis (BGP) has been shown to have immunomodulatory and antitumor properties in vitro and in vivo. We selected the caffeic acid phenethyl ester (CAPE), which is one of the components of BGP, as a candidate molecule for further studies regarding the antineoplastic activity. Cytotoxicity induced by Brazilian green propolis alcoholic extract (BGPAE) and CAPE in the cell lineages of acute promyelocytic leukemia (APL) (NB4 and NB4R2), and bone marrow cells from patients with acute myeloid leukemia (AML) at diagnosis (primary cells) was evaluated by annexin V/propidium iodide staining and analyzed by flow cytometry. BGPAE and CAPE showed antiproliferative and apoptotic activity and this effect was dose and time dependent (Table 1 and 2). Accordingly, in primary cells, CAPE (32μg/ml) for 24 hours significantly increased the percentage of apoptotic cells compared to control. The median (25 and 75 percentiles) for the percentage of apoptotic cells was 17.32% (13-27%) in control and 37% (24-52%) in CAPE, p-value = 0.0008 The cell cycle was analysed by flow cytometry using propidium iodide staining and demonstrated that CAPE blocked the cell cycle at G2/M. In addition, CAPE induced activation of caspases 3 and 9, as determined by Western blot, thus suggesting the involvement of the intrinsic pathway of apoptosis. The modulation of gene expression induced by CAPE in NB4 was determined by microarray using the CodeLink Uniset Human I bioarray (Amersham/GE) and analyzing 10,000 genes. Adopting a False Discovery Rate of 4.72% and using the Significance Analysis of Miroarray (SAM) software, we detected an increased expression of: negative mediators of cell cycle, including the dual specific phosphatases CDC14A, CDC14B and the cyclin-dependent kinase (CDK) inhibitor CDKN1A (p21/CIP1); protein phosphatases; chemokines and molecules associated with signaling by G protein. Furthermore, CAPE induced a decrease in gene expression of: positive mediators of cell cycle (including CDK4 and CCNA2); genes related to the “spliceosome” and protein translation. The most important differences in gene expression were confirmed by real time PCR using Taqman® technology (Applied Biosystems). Based on the microarray analysis, we decided to further study the production of IL-8 induced by CAPE in NB4 cells. Using an ELISA assay, it was detected a time dependent increase in the production of IL-8 at CAPE concentrations of 16 and 32μg/ml that was significantly higher than in controls at 12 and 24h of treatment. In conclusion, our results demonstrate that CAPE was able to block the cell cycle and induce apoptosis in APL cells, and induce the production of IL-8 ALP cells.Table 1Effective doses 50% (ED-50) of BGPAE (g/ml) for inhibition of proliferation and induction of apoptosis in NB4 and NB4R2 cell lines in 24, 48 and 72 hours of treatmenttimeED-50CI 95% for DE-50ProliferationNB424h63,7251,39 + 79,0048h43,0037,20 – 49,7072h33,5130,93 – 36,30NB4R224h30,2825,42 – 36,0648h50,6146,52 – 55,0672h50,4045,15 – 56,27ApoptosisNB424h214,2204,2 – 224,648h201,0189,0 – 213,872h185,4172,2 – 199,5NB4R224h139,0132,7 – 145,548h117,3112,3 – 122,572h101,595,18 – 108,2Table 2Effective doses 50% (ED-50) of CAPE (μg/ml) for inhibition of proliferation and induction of apoptosis in NB4 and NB4R2 cell lines in 24, 48 and 72 hours of treatmenttimeED-50CI 95% for DE-50ProliferationNB424h8,1066,977 – 9,41848h2,9452,571 – 3,37372h1,6481,451 – 1,872NB4R224h7,0296,209 – 7,95648h4,3424,017 – 4,69472h3,2143,052 – 3,384ApoptosisNB424h51,8144,67 – 60,1048h31,8828,71 – 35,3972h21,3818,99 – 24,07NB4R224h68,5858,66 – 80,1848h34,9230,39 – 40,1472h27,0224,26 – 30,09 Disclosures:No relevant conflicts of interest to declare.

Prognostic significance of CDKN2A (p16) promoter methylation and loss of expression in 902 colorectal cancers: Cohort study and literature review

A cyclin-dependent kinase inhibitor CDKN2A (p16/Ink4a) is a tumor suppressor and upregulated in cellular senescence. CDKN2A promoter methylation and gene silencing are associated with the CpG island methylator phenotype (CIMP) in colon cancer. However, prognostic significance of CDKN2A methylation or loss of CDKN2A (p16) expression independent of CIMP status remains uncertain. Using a database of 902 colorectal cancers in 2 independent cohort studies (the Nurses' Health Study and the Health Professionals Follow-up Study), we quantified CDKN2A promoter methylation and detected hypermethylation in 269 tumors (30%). By immunohistochemistry, we detected loss of CDKN2A (p16) expression in 25% (200/804) of tumors. We analyzed for LINE-1 hypomethylation and hypermethylation at 7 CIMP-specific CpG islands (CACNA1G, CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1); microsatellite instability (MSI); KRAS, BRAF and PIK3CA mutations; and expression of TP53 (p53), CTNNB1 (β-catenin), CDKN1A (p21), CDKN1B (p27), CCND1 (cyclin D1), FASN (fatty acid synthase) and PTGS2 (cyclooxygenase-2). CDKN2A promoter methylation and loss of CDKN2A (p16) were associated with shorter overall survival in univariate Cox regression analysis [hazard ratio (HR): 1.36, 95% CI: 1.10-1.66, p = 0.0036 for CDKN2A methylation; HR: 1.30, 95% CI: 1.03-1.63, p = 0.026 for CDKN2A (p16) loss] but not in multivariate analysis that adjusted for clinical and tumor variables, including CIMP, MSI and LINE-1 methylation. Neither CDKN2A promoter methylation nor loss of CDKN2A (p16) was associated with colorectal cancer-specific mortality in uni- or multivariate analysis. Despite its well-established role in carcinogenesis, CDKN2A (p16) promoter methylation or loss of expression in colorectal cancer is not independently associated with patient prognosis.

Runx1 Directly Promotes Proliferation of Hair Follicle Stem Cells and Epithelial Tumor Formation in Mouse Skin

Runx1/AML1 is a transcription factor implicated in tissue stem cell regulation and belongs to the small Runx family of cancer genes. In the hair follicle (HF), Runx1 epithelial deletion in morphogenesis impairs normal adult hair homeostasis (cycle) and blocks adult hair follicle stem cells (HFSCs) in quiescence. Here, we show that these effects are overcome later in adulthood. By deleting Runx1 after the end of morphogenesis, we demonstrate its direct role in promoting anagen onset and HFSC proliferation. Runx1 deletion resulted in cyclin-dependent kinase inhibitor Cdkn1a (p21) upregulation. Interfering with Runx1 function in cultured HFSCs impaired their proliferation and normal G(0)/G1 and G(1)/S cell cycle progression. The proliferation defect could be rescued by Runx1 readdition or by p21 deletion. Chemically induced skin tumorigenesis in mice turned on broad Runx1 expression in regions of the skin epithelium, papillomas, and squamous cell carcinomas. In addition, it revealed reduced rates of tumor formation in the absence of Runx1 that were accompanied by decreased epithelial levels of phospho-Stat3. Runx1 protein expression was similar in normal human and mouse hair cycles. We propose that Runx1 may act as a skin oncogene by directly promoting proliferation of the epithelial cells.

TGF-β Induces Growth Arrest in Burkitt Lymphoma Cells via Transcriptional Repression of E2F-1

Transforming growth factor-beta (TGF-beta) is a potent regulator of tissue homeostasis and can act as both a tumor suppressor and a tumor promoter. The ability to induce cell cycle arrest is a major component of the tumor suppressor function of TGF-beta. Lung, mammary, and skin epithelial cells exhibit a common minimal cytostatic program in response to TGF-beta signaling involving the repression of the growth-promoting factors c-MYC, Id1, Id2, and Id3. Loss of c-MYC expression is a pivotal event in this process, resulting in derepression of the cyclin-dependent kinase inhibitors CDKN1A (p21) and CDKN2B (p15) and ultimately leading to growth arrest. It is not clear, however, which responses are necessary for TGF-beta-mediated growth arrest in other cell types. Here, in human Burkitt lymphoma cells transformed by deregulated c-MYC expression, we demonstrate that efficient TGF-beta-induced cytostasis can occur despite both maintenance of c-MYC levels and a lack of p21 and p15 induction. TGF-beta treatment also results in induction, rather than repression, of Id1 and Id2 expression. In this context, growth arrest correlates with transcriptional repression of E2F-1, and overexpression of E2F-1 in Burkitt lymphoma cells largely overcomes the TGF-beta-mediated G(1) arrest phenotype. These data indicate that deregulation of c-MYC in lymphoma cells does not overcome the tumor suppressor function of TGF-beta and that repression of E2F-1 transcription is sufficient for the efficient induction of cytostasis.

Combined Analysis of Valproic Acid Induced MicroRNA and Gene Expression Changes in Acute Myeloid Leukemia.

Inhibitors of histone deacetylases (HDACs) like valproic acid (VPA) display activity in murine leukemia models and induce apoptosis and myeloid differentiation of acute myeloid leukemia (AML) blasts. While recently several studies examined the underlying VPA-mediated mechanisms, until today not many genes have been identified whose expression is altered by VPA treatment. Recently, microRNAs (miRs), a novel abundant class of negative gene regulators, have been shown to control a wide range of biological functions such as proliferation, differentiation and apoptosis by either translational repression or by mRNA cleavage or miR-mediated decay of the respective target mRNA. Furthermore, deregulated miR expression has been associated with various human cancers including leukemia. This led us to investigate whether VPA treatment of AML cells affects miR-expression which in turn might influence the level of miR target genes involved in VPA effects. First, we identified an in vitro miR VPA-response signature by profiling miR expression in 4 different myeloid leukemia cell lines following 48 hours of VPA treatment (Ambion microarray platform comprising 281 human miRs). In parallel, we profiled gene expression by using both cDNA microarrays and Affymetrix U133Aplus2.0 GeneChips. 13 miRs were found to be differentially expressed, 10 miRs were down-regulated and 3 miRs were up-regulated by VPA. Gene expression profiling revealed several hundred differentially regulated genes containing some known VPA influenced targets like e.g. cyclin-dependent kinase inhibitor CDKN1A coding for p21. To correlate miR and gene expression, we next searched for an enrichment of putative miR target genes of the VPA-regulated miRs in the VPA-induced gene expression pattern. Interestingly, there were several candidates for which miR expression in response to VPA inversely correlated with gene expression of the respective targets. These included genes involved in DNA damage checkpoint like e.g. CHEK1 which was found to be down-regulated in response to VPA and which is a predicted target of miR-15a and miR-16, both found to be up-regulated by VPA treatment. In addition, potential miR-targets included genes known to be regulated by HDAC inhibitors in cancer cells like e.g. the homeobox gene HOXA1 found to be up-regulated in response to VPA and being a putative target of miR-99a, found to be down-regulated by VPA. Our study is the first to show that VPA treatment significantly affects expression levels of several miRs in myeloid cell lines, and based on the correlation of VPA-induced miR and gene expression patterns we could identify putative miR-targets that included genes with tumorigenic relevance. While it remains to be determined whether VPA-induced miR-mediated mRNA cleavage or decay of the respective target mRNAs is involved in leukemogenesis, our data nevertheless provide new insights into VPA-induced mechanisms of myeloid differentiation and into deregulated miR expression in leukemia.

Quantifying a bystander response following microbeam irradiation using single-cell RT-PCR analyses

There is growing recognition that the effects of ionizing radiation may extend to more than those cells that directly suffer damage to DNA in the cell nucleus. Data from several investigators have indicated that cells neighboring those that are irradiated also demonstrate several responses seen in hit cells--the so-called bystander effect. The microbeam facility at the Center for Radiological Research is particularly well suited for the study of this bystander effect, since it has the ability to place known numbers of charged particles (protons or alpha-particles at LETs from 20 to 180 KeV/microm) at defined positions relative to individual cells. That is, some known fraction of cells in a population can be irradiated through the nucleus, or the cytoplasm or even adjacent to cells through the media. Therefore, using the microbeam it is possible to examine individual cell responses in both hit and nonhit cells in the same population. Alterations in the cyclin-dependent kinase inhibitor CDKN1a (p21/Cip1/WAF1) were quantified at the mRNA level in single normal human fibroblasts following precise delivery of 0 or 10 alpha-particles per cell at 90 KeV/microm to 50% of cells in a population. Semiquantitative RT-PCR of individual hit cells demonstrated increases in the levels of CDKN1A message that followed the kinetics previously described for irradiated populations. Furthermore, nonhit bystander cells also showed increased (though lesser) levels of CDKN1a message. Data presented here demonstrate the power of this approach, which combines the ability of the microbeam to irradiate specific cells in a population and the ability to quantify the response to the irradiation in individual targeted and bystander cells.

Ergosterol peroxide from an edible mushroom suppresses inflammatory responses in RAW264.7 macrophages and growth of HT29 colon adenocarcinoma cells

5alpha,8alpha-Epidioxy-22E-ergosta-6, 22-dien-3beta-ol (ergosterol peroxide) is a major antitumour sterol produced by edible or medicinal mushrooms. However, its molecular mechanism of action has yet to be determined. Here, we examine the anticancer and anti-inflammatory effects of ergosterol peroxide. After treating RAW264.7 macrophages with LPS and purified ergosterol peroxide or ergosterol, we determined LPS-induced inflammatory cytokines, nuclear DNA binding activity of transcription factors and phosphorylation of MAP kinases (MAPKs). HT29 colorectal adenocarcinoma cells were treated with ergosterol peroxide for 5 days. To investigate the antitumour properties of ergosterol peroxide, we performed DNA microarray and RT-PCR analyses and determined the reactive oxygen species (ROS) in HT29 cells. Ergosterol peroxide suppressed LPS-induced TNF-alpha secretion and IL-1alpha/beta expression in RAW264.7 cells. Ergosterol peroxide and ergosterol suppressed LPS-induced DNA binding activity of NF-kappaB and C/EBPbeta, and inhibited the phosphorylation of p38, JNK and ERK MAPKs. Ergosterol peroxide down-regulated the expression of low-density lipoprotein receptor (LDLR) regulated by C/EBP, and HMG-CoA reductase (HMGCR) in RAW264.7 cells. In addition, ergosterol peroxide showed cytostatic effects on HT29 cells and increased intracellular ROS. Furthermore, ergosterol peroxide induced the expression of oxidative stress-inducible genes, and the cyclin-dependent kinase inhibitor CDKN1A, and suppressed STAT1 and interferon-inducible genes. Our results suggest that ergosterol peroxide and ergosterol suppress LPS-induced inflammatory responses through inhibition of NF-kappaB and C/EBPbeta transcriptional activity, and phosphorylation of MAPKs. Moreover, ergosterol peroxide appears to suppress cell growth and STAT1 mediated inflammatory responses by altering the redox state in HT29 cells.

In Vitro and In Vivo Monitoring of Valproic Acid Effects on Gene Expression Signatures in Adult Acute Myeloid Leukemia.

Inhibitors of histone deacetylases (HDACIs) like valproic acid (VPA) display activity in murine leukemia models, and induce tumor-selective cytoxicity against blasts from patients with acute myeloid leukemia (AML). However, despite of the existing knowledge of the potential function of HDACIs, there remain many unsolved questions especially regarding the factors that determine whether a cancer cell undergoes cell cycle arrest, differentiation, or death in response to HDACIs. Furthermore, there is still limited data on HDACIs effects in vivo, as well as HDACIs function in combination with standard induction chemotherapy, as most studies evaluated HDACIs as single agent in vitro. Thus, our first goal was to determine a VPA response signature in different myeloid leukemia cell lines in vitro, followed by an in vivo analysis of VPA effects in blasts from adult de novo AML patients entered within two randomized multicenter treatment trials of the German-Austrian AML Study Group. To define an VPA in vitro “response signature” we profiled gene expression in myeloid leukemia cell lines (HL-60, NB-4, HEL-1, CMK and K-562) following 48 hours of VPA treatment by using DNA Microarray technology. In accordance with previous studies in vitro VPA treatment of myeloid cell lines induced the expression of the cyclin-dependent kinase inhibitors CDKN1A and CDKN2D coding for p21 and p19, respectively. Supervised analyses revealed many genes known to be associated with a G1 arrest. In all cell lines except for CMK we examined an up-regulation of TNFSF10 coding for TRAIL, as well as differential regulation of other genes involved in apoptosis. Furthermore, gene set enrichment analyses showed a significant down-regulation of genes involved in DNA metabolism and DNA repair. Next, we evaluated the VPA effects on gene expression in AML samples collected within the AMLSG 07-04 trial for younger (age<60yrs) and within the AMLSG 06-04 trial for older adults (age>60yrs), in which patients are randomized to receive standard induction chemotherapy (idarubicine, cytarabine, and etoposide = ICE) with or without concomitant VPA. We profiled gene expression in diagnostic AML blasts and following 48 hours of treatment with ICE or ICE/VPA. First results from our ongoing analysis of in vivo VPA treated samples are in accordance with our cell line experiments as e.g. we also see an induction of CDKN1A expression. However, the picture observed is less homogenous as concomitant administration of ICE, as well as other factors, like e.g. VPA serum levels, might substantially influence the in vivo VPA response. Nevertheless, our data are likely to provide new insights into the VPA effect in vivo, and this study may proof to be useful to predict AML patients likely to benefit from VPA treatment. To achieve this goal, we are currently analyzing additional samples, and we are planning to correlate gene expression findings with histone acetylation status, VPA serum levels, cytogenetic, and molecular genetic data.

Neural tube development requires the cooperation of p53‐ and Gadd45a‐associated pathways

Numerous genetically engineered mouse models for neural tube defects (NTDs) exist, and some of the implicated proteins are functionally related. For example, the growth arrest and DNA damage-inducible protein Gadd45a and tumor suppressor p53 are functionally similar, and both are involved in neural tube development (Gadd45a- and Trp53-null embryos show low levels of exencephaly). To assess their roles in neural tube development, we generated double-null mice from Gadd45a- and Trp53-null mice, as well as from cyclin-dependent kinase inhibitor (Cdkn1a) (p21)-null and xeroderma pigmentosum group C (XPC)-null mice that do not show spontaneous exencephaly. Gadd45a-, Trp53-, Cdkn1a-, and XPC-null mice were crossed to generate several double-null mouse models. Embryos (embryonic day [ED] 16-18) from the single- and double-null crosses were scored for NTDs. Deletion of both Gadd45a and Trp53 in mice increased exencephaly frequencies compared to the deletion of either single gene (34.0% in Gadd45a/Trp53-null compared to 8.4% and 9.1% in the Gadd45a- and Trp53-null embryos, respectively). Furthermore, although deletion of another p53-regulated gene, Cdkn1a, is not associated with exencephaly, in conjunction with Gadd45a deletion, the exencephaly frequencies are increased (30.5% in the Gadd45a/Cdkn1a-null embryos) and are similar to those in the Gadd45a/Trp53-null embryos. Although XPC deletion increased exencephaly frequencies in Trp53-null embryos, XPC deletion did not increase the exencephaly frequencies in Gadd45a-null embryos. The increased genetic liability to exencephaly in the Gadd45a/Trp53- and Gadd45a/Cdkn1a-null embryos may be related to the disruption of multiple cellular pathways associated with Gadd45a and p53.

Gene expression during sex determination reveals a robust female genetic program at the onset of ovarian development

The primary event in mammalian sexual development is the differentiation of the bipotential gonads into either testes or ovaries. Our understanding of the molecular pathways specifying gonadal differentiation is still incomplete. To identify the initial molecular changes accompanying gonadal differentiation in mice, we have performed a large-scale transcriptional analysis of XX and XY Sf1-positive gonadal cells during sex determination. In both male and female genital ridges, a robust genetic program is initiated pre-dating the first morphological changes of the differentiating gonads. Between E10.5 and E13.5, 2306 genes were expressed in a sex-specific manner in the somatic compartment of the gonads; 1223 were overexpressed in XX embryos and 1083 in XY embryos. Although sexually dimorphic genes were scattered throughout the mouse genome, we identified chromosomal regions hosting clusters of genes displaying similar expression profiles. The cyclin-dependent kinase inhibitors Cdkn1a and Cdkn1c are overexpressed in XX gonads at E11.5 and E12.5, suggesting that the increased proliferation of XY gonads relative to XX gonads may result from the overexpression of cell cycle inhibitors in the developing ovaries. These studies define the major characteristics of testicular and ovarian transcriptional programs and reveal the richness of signaling processes in differentiation of the bipotential gonads into testes and ovaries.

Retinoic acid induces CDK inhibitors and growth arrest specific (Gas) genes in neural crest cells

Retinoic acid (RA), the active metabolite of vitamin A, regulates cellular growth and differentiation during embryonic development. In excess, this vitamin is also highly teratogenic to animals and humans. The neural crest is particularly sensitive to RA, and high levels adversely affect migration, proliferation and cell death. We investigated potential gene targets of RA associated with neural crest proliferation by determining RA-mediated changes in gene expression over time, using microarrays. Statistical analysis of the top ranked RA-regulated genes identified modest changes in multiple genes previously associated with cell cycle control and proliferation including the cyclin-dependent kinase inhibitors Cdkn1a (p21), Cdkn2b (p15(INK4b)), and Gas3/PMP22. The expression of p21 and p15(INK4b) contribute to decreased proliferation by blocking cell cycle progression at G1-S. This checkpoint is pivotal to decisions regulating proliferation, apoptosis, or differentiation. We have also confirmed the overexpression of Gas3/PMP22 in RA-treated neural crests, which is associated with cytoskeletal changes and increased apoptosis. Our results suggest that increases in multiple components of diverse regulatory pathways have an overall cumulative effect on cellular decisions. This heterogeneity contributes to the pleiotropic effects of RA, specifically those affecting proliferation and cell death.

Inhibition of normal human lung fibroblast growth by beryllium

Inhalation of particulate beryllium (Be) and its compounds causes chronic Be disease (CBD) in a relatively small subset (∼1–6%) of exposed individuals. Hallmarks of this pulmonary disease include increases in several cell types, including lung fibroblasts, that contribute to the fibrotic component of the disorder. In this regard, enhancements in cell proliferation appear to play a fundamental role in CBD development and progression. Paradoxically, however, some existing evidence suggests that Be actually has antiproliferative effects. In order to gain further information about the effects of Be on cell growth, we: (1) assessed cell proliferation and cell cycle effects of low concentrations of Be in normal human diploid fibroblasts, and (2) investigated the molecular pathway(s) by which the cell cycle disturbing effects of Be may be mediated. Treatment of human lung and skin fibroblasts with Be added in the soluble form of BeSO 4 (0.1–100 μM) caused inhibitions of their growth in culture in a concentration-dependent manner. Such growth inhibition was found to persist, even after cells were further cultured in Be 2+-free medium. Flow cytometric analyses of cellular DNA labeled with the DNA-binding fluorochrome DAPI revealed that Be causes a G 0–G 1/pre-S phase arrest. Western blot analyses indicated that the Be-induced G 0–G 1/pre-S phase arrest involves elevations in TP53 (p53) and the cyclin-dependent kinase inhibitor CDKN1A (p21 Waf-1,Cip1). That Be at low concentrations inhibits the growth of normal human fibroblasts suggests the possibility of the existence of abnormal cell cycle inhibitory responses to Be in individuals who are sensitive to the metal and ultimately develop CBD.

Identification and mapping of swine cyclin-dependent kinase inhibitor CDKN2A and CDKN2B exon 2 sequences

We have isolated the swine homologs of human CDKN2A and CDKN2B exon 2 sequences. As in the human and mouse genomes, the exon 2 sequences of these two genes present a high level of sequence homology and are tightly linked. Using fluorescence in situ hybridization, we have mapped swine CDKN2A and CDKN2B to chromosome 1q25. This confirms the comparative mapping data among man, mouse, and swine, showing a conserved synteny among chromosome segments 9p21, 4C3-C6, and 1q25, respectively.

Role of the cyclin-dependent kinase inhibitor CDKN2A in familial melanoma.

Approximately 8 to 12% of melanoma appears to be inherited in an autosomal dominant form. Although most early stage melanomas can be treated successfully by simple surgical excision, patients with advanced disease are rarely cured even with aggressive chemotherapy and/or immunotherapy. There is now compelling evidence that germline mutations of the CDKN2A gene on chromosome 9p21 predispose to melanoma in a subset of melanoma-prone families. In this article the evidence for the role of CDKN2A in the genesis of familial melanoma is reviewed and the implications of genetic testing in families with this disease are discussed. The identification and subsequent surveillance of unaffected individuals who have a genetic predisposition to melanoma may lead to the detection of early (curable) melanomas and to a reduction in mortality.