In vitro phosphorylation of BRCA2 by the checkpoint kinase CHEK2.

Germline mutations in both BRCA2 and CHEK2 are associated with an increased risk for male breast cancer. To search for potential interactions between the products of these breast cancer susceptibility genes, we undertook systematic mapping of BRCA2 for potential phosphorylation sites by CHEK2. In vitro kinase assays and mass spectrometric analysis identified a 50 amino-acid fragment within the N-terminus of BRCA2 potentially targeted by CHEK2, containing two major phosphopeptides. Inducible overexpression of this peptide, but not a derivative with mutated phosphorylation sites, leads to increased chromosome fragmentation and suppression of cellular proliferation. These results suggest a link between CHEK2 and BRCA2 pathways, which may contribute to the spectrum of cancers associated with germline CHEK2 mutations.

Molecular targeted therapy of lung cancer: EGFR mutations and response to EGFR inhibitors.

Somatic mutations within the kinase domain of the epidermal growth factor receptor (EGFR) are present in approximately 10% of non-small-cell lung cancer (NSCLC), with an increased frequency in adenocarcinomas arising in nonsmokers, women, and individuals of Asian ethnicity. These mutations lead to altered downstream signaling by the receptor and appear to define a subset of NSCLC characterized by "oncogene addiction" to the EGFR pathway, which displays dramatic responses to the reversible tyrosine kinase inhibitors gefitinib and erlotinib. The rapid acquisition of drug resistance in most cases, either through mutation of the "gateway" residue in the EGFR kinase domain or by alternative mechanisms, appears to limit the impact on patient survival. Irreversible inhibitors of EGFR display continued effectiveness in vitro against cells with acquired resistance and are now undergoing genotype-directed clinical trials. The molecular and clinical insights derived from targeting EGFR in NSCLC offer important lessons for the broader application of targeted therapeutic agents in solid tumors.

WT1 regulates the expression of the major glomerular podocyte membrane protein Podocalyxin.

The WT1 tumor suppressor gene encodes a zinc finger transcription factor expressed in differentiating glomerular podocytes. Complete inactivation of WT1 in the mouse leads to failure of mesenchymal induction and renal agenesis, an early developmental phenotype that prevents analysis of subsequent stages in glomerular differentiation [1]. In humans with Denys-Drash Syndrome, a heterozygous germline mutation in WT1 is associated with specific defects in glomeruli and an increased risk for developing Wilms Tumor [2,3]. WT1 target genes implicated in cell cycle regulation and cellular proliferation have been proposed [4], but the link between WT1 function and glomerular differentiation is unexplained. Here, we show that inducible expression of WT1 in rat embryonic kidney cell precursors leads to the induction of endogenous Podocalyxin, the major structural membrane protein of glomerular podocytes, which is implicated in the maintenance of filtration slits. Binding of WT1 to conserved elements within the Podocalyxin gene promoter results in potent transcriptional activation, and the specific expression pattern of Podocalyxin in the developing kidney mirrors that of WT1 itself. These observations support a role for WT1 in the specific activation of a glomerular differentiation program in renal precursors and provide a molecular basis for the glomerulonephropathy that is characteristic of Denys-Drash Syndrome.

Destabilization of CHK2 by a missense mutation associated with Li-Fraumeni Syndrome.

Li Fraumeni Syndrome (LFS) is a multicancer phenotype, most commonly associated with germ-line mutations in TP53. In a kindred with LFS without an inherited TP53 mutation, we have previously reported a truncating mutation (1100delC) in CHK2, encoding a kinase that phosphorylates p53 on Ser(20). Here, we describe a CHK2 missense mutation (R145W) in another LFS family. This mutation destabilizes the encoded protein, reducing its half-life from >120 min to 30 min. This effect is abrogated by treatment of cells with a proteosome inhibitor, suggesting that CHK2(R145W) is targeted through this degradation pathway. Both 1100delC and R145W germ-line mutations in CHK2 are associated with loss of the wild-type allele in the corresponding tumor specimens, and neither tumor harbors a somatic TP53 mutation. Our observations support the functional significance of CHK2 mutations in rare cases of LFS and suggest that such mutations may substitute for inactivation of TP53.

Loss of imprinting of insulin-like growth factor-II (IGF2) gene in distinguishing specific biologic subtypes of Wilms tumor.

BACKGROUND: Loss of imprinting (LOI) of the insulin-like growth factor-II (IGF2) gene, an epigenetic alteration associated with expression of the normally silent maternal allele, was observed first in Wilms tumor. Although LOI has subsequently been detected in most adult tumors, the biologic role of LOI in cancer remains obscure. We analyzed the imprinting status of Wilms tumors with respect to pathologic subtype, stage, and patient's age at diagnosis and examined the expression of genes potentially affected by LOI. METHODS: Of 60 Wilms tumors examined, 25 were informative for an ApaI polymorphism in the IGF2 gene, allowing analysis of allele-specific gene expression, and could be classified by pathologic subtype. Gene expression was measured quantitatively by real-time polymerase chain reaction, and pathologic analysis was blinded for genetic status. All statistical tests were two-sided. RESULTS: We observed LOI of IGF2 in nine (90%) of 10 Wilms tumors classified as having a pathologic subtype associated with a later stage of renal development and in only one (6.7%) of 15 Wilms tumors with a pathologic subtype associated with an earlier stage of renal development (P< .001). LOI was associated with a 2.2-fold increase (95% confidence interval [CI] = 1.6-fold to 3.1-fold) in IGF2 expression (P< .001). Children whose Wilms tumors displayed LOI of IGF2 were statistically significantly older at diagnosis (median = 65 months; interquartile range [IQR] = 47-83 months) than children whose tumors displayed normal imprinting (median = 24 months; IQR = 13-35 months; P< .001). CONCLUSIONS: These data demonstrate a clear relationship between LOI and altered expression of IGF2 in Wilms tumors and provide a molecular basis for understanding the divergent pathogenesis of this cancer. Analysis of LOI could provide a valuable molecular tool for the classification of Wilms tumor.

BRCA1 and GADD45 mediated G2/M cell cycle arrest in response to antimicrotubule agents.

BRCA1 is a tumour suppressor gene implicated in the predisposition to early onset breast and ovarian cancer. We have generated cell lines with inducible expression of BRCA1 to evaluate its role in mediating the cellular response to various chemotherapeutic drugs commonly used in the treatment of breast and ovarian cancer. Induction of BRCA1 in the presence of Taxol and Vincristine resulted in a dramatic increase in cell death; an effect that was preceded by an acute arrest at the G2/M phase of the cell cycle and which correlated with BRCA1 mediated induction of GADD45. A proportion of the arrested cells were blocked in mitosis suggesting activation of both a G2 and a mitotic spindle checkpoint. In contrast, no specific interaction was observed between BRCA1 induction and treatment of cells with a range of DNA damaging agents including Cisplatin and Adriamycin. Inducible expression of GADD45 in the presence of Taxol induced both G2 and mitotic arrest in these cells consistent with a role for GADD45 in contributing to these effects. Our results support a role for both BRCA1 and GADD45 in selectively regulating a G2/M checkpoint in response to antimicrotubule agents and raise the possibility that their expression levels in cells may contribute to the toxicity observed with these compounds.

Archipelago regulates Cyclin E levels in Drosophila and is mutated in human cancer cell lines.

During Drosophila development and mammalian embryogenesis, exit from the cell cycle is contingent on tightly controlled downregulation of the activity of Cyclin E-Cdk2 complexes that normally promote the transition from G1 to S phase. Although protein degradation has a crucial role in downregulating levels of Cyclin E, many of the proteins that function in degradation of Cyclin E have not been identified. In a screen for Drosophila mutants that display increased cell proliferation, we identified archipelago, a gene encoding a protein with an F-box and seven tandem WD (tryptophan-aspartic acid) repeats. Here we show that archipelago mutant cells have persistently elevated levels of Cyclin E protein without increased levels of cyclin E RNA. They are under-represented in G1 fractions and continue to proliferate when their wild-type neighbours become quiescent. The Archipelago protein binds directly to Cyclin E and probably targets it for ubiquitin-mediated degradation. A highly conserved human homologue is present and is mutated in four cancer cell lines including three of ten derived from ovarian carcinomas. These findings implicate archipelago in developmentally regulated degradation of Cyclin E and potentially in the pathogenesis of human cancers.

Cascades of transcriptional induction during human lymphocyte activation.

Lymphocyte activation is known to be associated with the induction of genes implicated in cytokine signaling and cellular proliferation. High-density microarrays offer the means to monitor global cellular expression profiles, temporal relationships between classes of transcripts, and alterations associated with human disease or immunosuppression. We sought to determine whether microarray analysis would accurately reflect the normal pattern of gene expression following human T cell activation, and whether the complex expression patterns identified could be analyzed to produce a functional profile of lymphocyte activation. We examined a time course of sequential expression profiles for 6,800 cellular transcripts in human lymphocytes activated with concanavalin A. Expression patterns were grouped using clustering analysis and validated using Northern blotting. Genes known to be induced following T cell activation were accurately identified, and the qualitative patterns of gene expression were well correlated between Northern and microarray analyses. Quantitative differences in gene expression levels were less well correlated between these two techniques. Expression profile analysis revealed the sequential induction of groups of functionally similar genes, whose temporal coregulation underscores known cellular events during T cell activation. This functional "fingerprint" of lymphocyte activation may prove useful for comparisons of lymphocyte responses under experimental conditions and in disease states.

BACH1, a novel helicase-like protein, interacts directly with BRCA1 and contributes to its DNA repair function.

BRCA1 interacts in vivo with a novel protein, BACH1, a member of the DEAH helicase family. BACH1 binds directly to the BRCT repeats of BRCA1. A BACH1 derivative, bearing a mutation in a residue that was essential for catalytic function in other helicases, interfered with normal double-strand break repair in a manner that was dependent on its BRCA1 binding function. Thus, BACH1/BRCA1 complex formation contributes to a key BRCA1 activity. In addition, germline BACH1 mutations affecting the helicase domain were detected in two early-onset breast cancer patients and not in 200 matched controls. Thus, it is conceivable that, like BRCA1, BACH1 is a target of germline cancer-inducing mutations.

The Wilms tumor suppressor WT1 directs stage-specific quiescence and differentiation of human hematopoietic progenitor cells.

WT1, a transcription factor implicated in both normal kidney differentiation and tumorigenesis, is also expressed in differentiating hematopoietic progenitors. Most human acute leukemias contain high levels of the wild-type transcript, while a minority have point mutations, raising the possibility that this tumor suppressor might have a paradoxical oncogenic effect in some hematopoietic cells. Using high titer retroviral infection, we demonstrate that WT1 triggers rapid growth arrest and lineage-specific differentiation in primary hematopoietic progenitors and differentiation-competent leukemia cell lines, while it induces cellular quiescence in a primitive subset of primary precursors. Growth arrest by WT1 is associated with induction of p21(CIP1), but expression of this cyclin-dependent kinase inhibitor alone is insufficient for either cellular differentiation or primitive cell preservation. The effects of WT1 are enhanced by co-expression of its naturally occurring isoforms, and are correlated with the physiological expression pattern of WT1 in vivo. Our observations suggest a role for WT1 in the differentiation of human hematopoietic cells, and provide a functional model that supports its capacity as a tumor suppressor in human acute leukemia.

A functional interaction with CBP contributes to transcriptional activation by the Wilms tumor suppressor WT1.

The Wilms tumor gene WT1 encodes a zinc finger transcription factor that is required for normal kidney development. WT1 was identified as a transcriptional repressor, based on its suppression of promoter reporters, but analysis of native transcripts using high density microarrays has uncovered transcriptional activation, rather than repression, of potential target genes. We report here that WT1 binds to the transcriptional coactivator CBP, leading to synergistic activation of a physiologically relevant promoter. The physical interaction between WT1 and CBP is evident in vitro and in vivo, and the two proteins are co-immunoprecipitated from embryonic rat kidney cells. The WT1-CBP association requires the first two zinc fingers of WT1 and the adenovirus 5 E1A-binding domain of CBP. Overexpression of this domain of CBP is sufficient to inhibit WT1-mediated transcriptional activation of a promoter reporter, as is co-transfection of E1A. Retrovirally driven expression of either the CBP fragment or of E1A in human hematopoietic cells suppresses the induction by WT1 of its endogenous target gene, p21(Cip1). These observations support a model of WT1 as a transcriptional activator of genes required for cellular differentiation.

Wilms tumor and the WT1 gene.

Wilms tumor or nephroblastoma is a pediatric kidney cancer arising from pluripotent embryonic renal precursors. Multiple genetic loci have been linked to Wilms tumorigenesis; positional cloning strategies have led to the identification of the WT1 tumor suppressor gene at chromosome 11p13. WT1 encodes a zinc finger transcription factor that is inactivated in the germline of children with genetic predisposition to Wilms tumor and in a subset of sporadic cancers. When present in the germline, specific heterozygous dominant-negative mutations are associated with severe abnormalities of renal and sexual differentiation, pointing to the essential role of WT1 for normal genitourinary development. The role of this tumor suppressor in normal organ-specific differentiation is also supported by the highly restricted temporal and spatial expression of WT1 in glomerular precursors of the developing kidney and by the failure of kidney development in wt1-null mice. Of two major alternative splicing products encoded by WT1, the (-KTS) isoform appears to mediate transcriptional activation of genes implicated in cellular differentiation, possibly also repressing proliferation-associated genes. The (+KTS) isoform, whose DNA-binding domain is disrupted by the insertion of three amino acids, may be involved in some aspect of mRNA processing. In addition to its function in genitourinary development, a role for WT1 in hematopoiesis is suggested by its aberrant expression and/or mutation in a subset of acute human leukemias. WT1 is also expressed in mesothelial cells; a specific oncogenic chromosomal translocation fusing the N-terminal domain of the Ewing sarcoma gene EWS to the three C-terminal zinc fingers of WT1 underlies desmoplastic small round cell tumor, an abdominal tumor thought to arise from the peritoneal lining. Understanding the distinct functional properties of WT1 isoforms and tumor-associated variants will provide unique insight into the link between normal organ-specific differentiation and malignancy.

Hemophagocytic lymphohistiocytosis due to germline mutations in SH2D1A, the X-linked lymphoproliferative disease gene.

The hemophagocytic lymphohistiocytoses (HLH) comprise a heterogeneous group of disorders characterized by dysregulated activation of T cells and macrophages. Although some patients with HLH harbor perforin gene mutations, the cause of the remaining cases is not known. The phenotype of HLH bears a strong resemblance to X-linked lymphoproliferative disease (XLP), an Epstein-Barr virus (EBV)-associated immunodeficiency resulting from defects in SH2D1A, a small SH2 domain-containing protein expressed in T lymphocytes and natural killer cells. Here it is shown that 4 of 25 male patients with HLH who were examined harbored germline SH2D1A mutations. Among these 4 patients, only 2 had family histories consistent with XLP. On the basis of these findings, it is suggested that all male patients with EBV-associated hemophagocytosis be screened for mutations in SH2D1A. Patients identified as having XLP should undergo genetic counseling, and be followed long-term for development of lymphoma and hypogammaglobulinemia.

Genetic susceptibility to breast cancer: HLA DQB*03032 and HLA DRB1*11 may represent protective alleles.

Tumors are believed to emerge only when immune surveillance fails. We wished to ascertain whether the failure to inherit putative protective alleles of HLA class II genes is linked to the development of breast cancer. We molecularly typed HLA DPB1, DQB1, DRB1, and DRB3 alleles in 176 Caucasian women diagnosed with early-onset breast cancer and in 215 ethnically matched controls. HLA DQB*03032 was identified in 7% of controls but in no patients with early-onset breast cancer (P = 0.0001). HLA DRB1*11 alleles were also significantly overrepresented (P < 0.0001) in controls (16. 3%) as compared with patients with early-onset breast cancer (3.5%). HLA DQB*03032 and HLA DRB1*11 alleles may have a protective role in human breast cancer.

Phenol sulfotransferases: hormonal regulation, polymorphism, and age of onset of breast cancer.

In recent years, significant effort has been made to identify genes that influence breast cancer risk. Because the high-penetrance breast cancer susceptibility genes BRCA1 and 2 play a role only in a small fraction of breast cancer cases, understanding the genetic risk of the majority of breast cancers will require the identification and analysis of several lower penetrance genes. The estrogen-signaling pathway plays a crucial role in the pathophysiology of breast cancer; therefore, polymorphism in genes involved in this pathway is likely to influence breast cancer risk. Our detailed analysis of gene expression profiles of estrogen- and 4-OH-tamoxifen-treated ZR75-1 breast cancer cells identified members of the sulfotransferase 1A (SULT1A) phenol sulfotransferase family as downstream targets of tamoxifen. On the basis of the induction of SULT1A by 4-OH-tamoxifen and the known inherited variability in SULT1A enzymatic activity, we hypothesized that polymorphism in sulfotransferase genes might influence the risk of breast cancer. Using an RFLP that distinguishes an arginine to histidine change in exon 7 of the SULT1A1 gene, we characterized SULT1A1 genotypes in relation to breast cancer risk. An analysis of 444 breast cancer patients and 227 controls revealed no effect of SULT1A1 genotype on the risk of breast cancer (P = 0.69); however, it did appear to influence the age of onset among early-onset affected patients (P = 0.04). Moreover, individuals with the higher activity SULT1A1*1 allele were more likely to have other tumors in addition to breast cancer (P = 0.004; odds ratio, 3.02; 95% confidence interval, 1.32, 8.09). The large number of environmental mutagens and carcinogens activated by sulfotransferases and the high frequency of the SULT1A1*1 allele in human populations warrants additional studies to address the role of SULT genes in human cancer.

Prevalence of germline truncating mutations in ATM in women with a second breast cancer after radiation therapy for a contralateral tumor.

Patients treated with conservative surgery and radiation therapy for early-stage breast cancer develop a contralateral breast cancer at a rate of approximately 0.75% per year. Ataxia-telangiectasia (AT) is an autosomal recessive disease that is characterized by increased sensitivity to ionizing radiation (IR) and cancer susceptibility. Epidemiologic studies have suggested that AT carriers, who comprise 1% of the population, may be at an increased risk for developing breast cancer, particularly after exposure to IR. To test this hypothesis, we analyzed blood samples from 57 patients who developed a contralateral breast cancer at least 6 months after completion of radiation therapy for an initial breast tumor. A cDNA-based truncation assay in yeast was used to test for heterozygous mutations in the ATM gene, which is responsible for AT. No mutations were detected. Our findings fail to support the hypothesis that AT carriers account for a significant fraction of breast cancer cases arising in women after exposure to radiation. Genes Chromosomes Cancer 27:124-129, 2000.

DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development.

The establishment of DNA methylation patterns requires de novo methylation that occurs predominantly during early development and gametogenesis in mice. Here we demonstrate that two recently identified DNA methyltransferases, Dnmt3a and Dnmt3b, are essential for de novo methylation and for mouse development. Inactivation of both genes by gene targeting blocks de novo methylation in ES cells and early embryos, but it has no effect on maintenance of imprinted methylation patterns. Dnmt3a and Dnmt3b also exhibit nonoverlapping functions in development, with Dnmt3b specifically required for methylation of centromeric minor satellite repeats. Mutations of human DNMT3B are found in ICF syndrome, a developmental defect characterized by hypomethylation of pericentromeric repeats. Our results indicate that both Dnmt3a and Dnmt3b function as de novo methyltransferases that play important roles in normal development and disease.

Heterozygous germline ATM mutations do not contribute to radiation-associated malignancies after Hodgkin's disease.

PURPOSE: The successful treatment of Hodgkin's disease has been associated with an increased incidence of secondary malignancies. To investigate whether genetic factors contribute to the development of secondary tumors, we collected family cancer histories and performed mutational analysis of the ataxia-telangiectasia (AT) gene, ATM, in a cohort of Hodgkin's disease survivors with secondary malignancies. ATM was chosen for evaluation because of the increased radiosensitivity of cells derived from AT patients and obligate heterozygotes and the epidemiologic observation that AT carriers are at increased risk for radiation-induced breast cancer. PATIENTS AND METHODS: Fifty-two patients who developed one or more neoplasms after treatment for Hodgkin's disease participated in this study. Personal and family histories of cancer were obtained through patient interviews and review of medical records. ATM mutational analysis was performed using a yeast-based protein truncation assay. RESULTS: Seventy-six secondary neoplasms were observed in this cohort of 52 Hodgkin's disease survivors, with 18 patients (35%) developing more than one secondary neoplasm. Positive family histories of cancer were present in 11 (21%) of 52 patients, compared with three (4%) of 68 Hodgkin's disease patients in a comparison cohort who did not develop secondary neoplasms (P =.008; Fisher's exact test). No germline ATM mutations were identified, resulting in an estimated AT carrier frequency in this population of 0% (90% confidence interval, 0% to 4%). CONCLUSION: Analysis of the number of tumors per individual and the family history of cancer in our cohort suggests that genetic factors may contribute to development of secondary neoplasms in a subset of Hodgkin's disease survivors. Mutational analysis, however, does not support a significant role for heterozygous truncating ATM mutations. Future studies evaluating other genes involved in DNA damage response pathways are warranted.