PIK3CA is implicated as an oncogene in ovarian cancer.

Ovarian cancer is the leading cause of death from gynecological malignancy and the fourth leading cause of cancer death among American women, yet little is known about its molecular aetiology. Studies using comparative genomic hybridization (CGH) have revealed several regions of recurrent, abnormal, DNA sequence copy number that may encode genes involved in the genesis or progression of the disease. One region at 3q26 found to be increased in copy number in approximately 40% of ovarian and others cancers contains PIK3CA, which encodes the p110alpha catalytic subunit of phosphatidylinositol 3-kinase (PI3-kinase). The association between PIK3CA copy number and PI3-kinase activity makes PIK3CA a candidate oncogene because a broad range of cancer-related functions have been associated with PI3-kinase mediated signalling. These include proliferation, glucose transport and catabolism, cell adhesion, apoptosis, RAS signalling and oncogenic transformation. In addition, downstream effectors of PI3-kinase, AKT1 and AKT2, have been found to be amplified or activated in human tumours, including ovarian cancer. We show here that PIK3CA is frequently increased in copy number in ovarian cancers, that the increased copy number is associated with increased PIK3CA transcription, p110alpha protein expression and PI3-kinase activity and that treatment with the PI3-kinase inhibitor LY294002 decreases proliferation and increases apoptosis. Our observations suggest PIK3CA is an oncogene that has an important role in ovarian cancer.

Quantitative mapping of amplicon structure by array CGH identifies CYP24 as a candidate oncogene.

We show here that quantitative measurement of DNA copy number across amplified regions using array comparative genomic hybridization (CGH) may facilitate oncogene identification by providing precise information on the locations of both amplicon boundaries and amplification maxima. Using this analytical capability, we resolved two regions of amplification within an approximately 2-Mb region of recurrent aberration at 20q13.2 in breast cancer. The putative oncogene ZNF217 (ref. 5) mapped to one peak, and CYP24 (encoding vitamin D 24 hydroxylase), whose overexpression is likely to lead to abrogation of growth control mediated by vitamin D, mapped to the other.

Tumour amplified kinase STK15/BTAK induces centrosome amplification, aneuploidy and transformation.

The centrosomes are thought to maintain genomic stability through the establishment of bipolar spindles during cell division, ensuring equal segregation of replicated chromosomes to two daughter cells. Deregulated duplication and distribution of centrosomes have been implicated in chromosome segregation abnormalities, leading to aneuploidy seen in many cancer cell types. Here, we report that STK15 (also known as BTAK and aurora2), encoding a centrosome-associated kinase, is amplified and overexpressed in multiple human tumour cell types, and is involved in the induction of centrosome duplication-distribution abnormalities and aneuploidy in mammalian cells. STK15 amplification has been previously detected in breast tumour cell lines and in colon tumours; here, we report its amplification in approximately 12% of primary breast tumours, as well as in breast, ovarian, colon, prostate, neuroblastoma and cervical cancer cell lines. Additionally, high expression of STK15 mRNA was detected in tumour cell lines without evidence of gene amplification. Ectopic expression of STK15 in mouse NIH 3T3 cells led to the appearance of abnormal centrosome number (amplification) and transformation in vitro. Finally, overexpression of STK15 in near diploid human breast epithelial cells revealed similar centrosome abnormality, as well as induction of aneuploidy. These findings suggest that STK15 is a critical kinase-encoding gene, whose overexpression leads to centrosome amplification, chromosomal instability and transformation in mammalian cells.

High resolution analysis of DNA copy number variation using comparative genomic hybridization to microarrays.

Gene dosage variations occur in many diseases. In cancer, deletions and copy number increases contribute to alterations in the expression of tumour-suppressor genes and oncogenes, respectively. Developmental abnormalities, such as Down, Prader Willi, Angelman and Cri du Chat syndromes, result from gain or loss of one copy of a chromosome or chromosomal region. Thus, detection and mapping of copy number abnormalities provide an approach for associating aberrations with disease phenotype and for localizing critical genes. Comparative genomic hybridization (CGH) was developed for genome-wide analysis of DNA sequence copy number in a single experiment. In CGH, differentially labelled total genomic DNA from a 'test' and a 'reference' cell population are cohybridized to normal metaphase chromosomes, using blocking DNA to suppress signals from repetitive sequences. The resulting ratio of the fluorescence intensities at a location on the 'cytogenetic map', provided by the chromosomes, is approximately proportional to the ratio of the copy numbers of the corresponding DNA sequences in the test and reference genomes. CGH has been broadly applied to human and mouse malignancies. The use of metaphase chromosomes, however, limits detection of events involving small regions (of less than 20 Mb) of the genome, resolution of closely spaced aberrations and linking ratio changes to genomic/genetic markers. Therefore, more laborious locus-by-locus techniques have been required for higher resolution studies. Hybridization to an array of mapped sequences instead of metaphase chromosomes could overcome the limitations of conventional CGH (ref. 6) if adequate performance could be achieved. Copy number would be related to the test/reference fluorescence ratio on the array targets, and genomic resolution could be determined by the map distance between the targets, or by the length of the cloned DNA segments. We describe here our implementation of array CGH. We demonstrate its ability to measure copy number with high precision in the human genome, and to analyse clinical specimens by obtaining new information on chromosome 20 aberrations in breast cancer.

Alagille syndrome is caused by mutations in human Jagged1, which encodes a ligand for Notch1.

Alagille syndrome is an autosomal dominant disorder characterized by abnormal development of liver, heart, skeleton, eye, face and, less frequently, kidney. Analyses of many patients with cytogenetic deletions or rearrangements have mapped the gene to chromosome 20p12, although deletions are found in a relatively small proportion of patients (< 7%). We have mapped the human Jagged1 gene (JAG1), encoding a ligand for the developmentally important Notch transmembrane receptor, to the Alagille syndrome critical region within 20p12. The Notch intercellular signalling pathway has been shown to mediate cell fate decisions during development in invertebrates and vertebrates. We demonstrate four distinct coding mutations in JAG1 from four Alagille syndrome families, providing evidence that it is the causal gene for Alagille syndrome. All four mutations lie within conserved regions of the gene and cause translational frameshifts, resulting in gross alterations of the protein product Patients with cytogenetically detectable deletions including JAG1 have Alagille syndrome, supporting the hypothesis that haploinsufficiency for this gene is one of the mechanisms causing the Alagille syndrome phenotype.

Actinin-associated LIM protein: identification of a domain interaction between PDZ and spectrin-like repeat motifs.

PDZ motifs are protein-protein interaction domains that often bind to COOH-terminal peptide sequences. The two PDZ proteins characterized in skeletal muscle, syntrophin and neuronal nitric oxide synthase, occur in the dystrophin complex, suggesting a role for PDZ proteins in muscular dystrophy. Here, we identify actinin-associated LIM protein (ALP), a novel protein in skeletal muscle that contains an NH2-terminal PDZ domain and a COOH-terminal LIM motif. ALP is expressed at high levels only in differentiated skeletal muscle, while an alternatively spliced form occurs at low levels in the heart. ALP is not a component of the dystrophin complex, but occurs in association with alpha-actinin-2 at the Z lines of myofibers. Biochemical and yeast two-hybrid analyses demonstrate that the PDZ domain of ALP binds to the spectrin-like motifs of alpha-actinin-2, defining a new mode for PDZ domain interactions. Fine genetic mapping studies demonstrate that ALP occurs on chromosome 4q35, near the heterochromatic locus that is mutated in fascioscapulohumeral muscular dystrophy.

ZAP-70 deficiency in an autosomal recessive form of severe combined immunodeficiency.

Protein tyrosine kinases (PTKs) play an integral role in T cell activation and differentiation. Defects in the Src-family PTKs in mice and in T cell lines have resulted in variable defects in thymic development and in T cell antigen receptor (TCR) signal transduction. Here, three siblings are described with an autosomal recessive form of severe combined immunodeficiency disease (SCID) in which ZAP-70, a non-Src PTK, is absent as a result of mutations in the ZAP-70 gene. This absence is associated with defects in TCR signal transduction, suggesting an important functional role for ZAP-70.

Differentiation of central nervous system neuronal cells by fibroblast-derived growth factor requires at least two signaling pathways: roles for Ras and Src.

To evaluate the role of mitogen-activated protein (MAP) kinase and other signaling pathways in neuronal cell differentiation by basic fibroblast-derived growth factor (bFGF), we used a conditionally immortalized cell line from rat hippocampal neurons (H19-7). Previous studies have shown that activation of MAP kinase kinase (MEK) is insufficient to induce neuronal differentiation of H19-7 cells. To test the requirement for MEK and MAP kinase (ERK1 and ERK2), H19-7 cells were treated with the MEK inhibitor PD098059. Although the MEK inhibitor blocked the induction of differentiation by constitutively activated Raf, the H19-7 cells still underwent differentiation by bFGF. These results suggest that an alternative pathway is utilized by bFGF for differentiation of the hippocampal neuronal cells. Expression in the H19-7 cells of a dominant-negative Ras (N17-Ras) or Raf (C4-Raf) blocked differentiation by bFGF, suggesting that Ras and probably Raf are required. Expression of dominant-negative Src (pcSrc295Arg) or microinjection of an anti-Src antibody blocked differentiation by bFGF in H19-7 cells, indicating that bFGF also signals through a Src kinase-mediated pathway. Although neither constitutively activated MEK (MEK-2E) nor v-Src was sufficient individually to differentiate the H19-7 cells, coexpression of constitutively activated MEK and v-Src induced neurite outgrowth. These results suggest that (i) activation of MAP kinase (ERK1 and ERK2) is neither necessary nor sufficient for differentiation by bFGF; (ii) activation of Src kinases is necessary but not sufficient for differentiation by bFGF; and (iii) differentiation of H19-7 neuronal cells by bFGF requires at least two signaling pathways activated by Ras and Src.

Extracellular signal-regulated kinase 7 (ERK7), a novel ERK with a C-terminal domain that regulates its activity, its cellular localization, and cell growth.

Mitogen-activated protein (MAP) kinases play distinct roles in a variety of cellular signaling pathways and are regulated through multiple mechanisms. In this study, a novel 61-kDa member of the MAP kinase family, termed extracellular signal-regulated kinase 7 (ERK7), has been cloned and characterized. Although it has the signature TEY activation motif of ERK1 and ERK2, ERK7 is not activated by extracellular stimuli that typically activate ERK1 and ERK2 or by common activators of c-Jun N-terminal kinase (JNK) and p38 kinase. Instead, ERK7 has appreciable constitutive activity in serum-starved cells that is dependent on the presence of its C-terminal domain. Interestingly, the C-terminal tail, not the kinase domain, of ERK7 regulates its nuclear localization and inhibition of growth. Taken together, these results elucidate a novel type of MAP kinase whereby interactions via its C-terminal tail, rather than extracellular signal-mediated activation cascades, regulate its activity, localization, and function.

Raf, but not MEK or ERK, is sufficient for differentiation of hippocampal neuronal cells.

To elucidate signal transduction pathways leading to neuronal differentiation, we have investigated a conditionally immortalized cell line from rat hippocampal neurons (H19-7) that express a temperature sensitive simian virus 40 large T antigen. Treatment of H19-7 cells with the differentiating agent basic fibroblast growth factor at 39 degrees C, the nonpermissive temperature for T function, resulted in the activation of c-Raf-1, MEK, and mitogen-activated protein (MAP) kinases (ERK1 and -2). To evaluate the role of Raf-1 in neuronal cell differentiation, we stably transfected H19-7 cells with v-raf or an oncogenic human Raf-1-estrogen receptor fusion gene (deltaRaf-1:ER). deltaRaf-1:ER transfectants in the presence of estradiol for 1 to 2 days expressed a differentiation phenotype only at the nonpermissive temperature. However, extended exposure of the deltaRaf-1:ER transfectants to estradiol or stable expression of the v-raf construct yielded cells that extended processes at the permissive as well as the nonpermissive temperature, suggesting that cells expressing the large T antigen are capable of responding to the Raf differentiation signal. deltaRaf-1:ER, MEK, and MAP kinase activities in the deltaRaf-1:ER cells were elevated constitutively for up to 36 h of estradiol treatment at the permissive temperature. At the nonpermissive temperature, MEK and ERKs were activated to a significantly lesser extent, suggesting that prolonged MAP kinase activation may not be sufficient for differentiation. To test this possibility, H19-7 cells were transfected or microinjected with constitutively activated MEK. The results indicate that prolonged activation of MEK or MAP kinases (ERK1 and -2) is not sufficient for differentiation of H19-7 neuronal cells and raise the possibility that an alternative signaling pathway is required for differentiation of H19-7 cells by Raf.

Neocarzinostatin-mediated DNA damage and repair in wild-type and repair-deficient Chinese hamster ovary cells.

The formation and repair of neocarzinostatin (NCS)-mediated DNA damage were examined in two strains of Chinese hamster ovary cells. The response in strain EM9, a mutant line selected for its sensitivity to ethyl methanesulfonate and shown to have a defect in the repair of X-ray-induced DNA breaks, was compared with that observed in the parental strain (AA8). The DNA strand breaks and their subsequent rejoining were measured using the method of elution of DNA from filters under either alkaline (for single-strand breaks), or nondenaturing conditions (for double-strand breaks). Colony survival assays showed that the mutant was more sensitive to the action of NCS than was the parental strain by a factor of approximately 1.5. Elution analyses showed that the DNA from both strains was damaged by NCS; the mutant displayed more damage than the parent under the same treatment conditions. Single-strand breaks were produced with a frequency of about 10 to 15 times the frequency of double-strand breaks. Both strains were able to rejoin both single-strand breaks and double-strand breaks induced by NCS treatment. The strand break data suggest that the difference in NCS-mediated cytotoxicity between EM9 and AA8 cells may be directly related to the enhanced production of DNA strand breaks in EM9. However, the fact that much higher doses of NCS were required in the DNA studies compared to the colony survival assays implies that either a small number of DNA breaks occur in a critical region of the genome, or that lesions other than DNA strand breaks are partly responsible for the observed cytotoxicity.

A candidate tumor suppressor gene in human breast cancers.

We have isolated a candidate gene (designated Brush-1) located at 13q12-q13, proximal to the retinoblastoma gene (RB1). Brush-1 codes for a 4.7-kilobase mRNA expressed at high levels in normal breast epithelium but drastically reduced in 6 of 13 breast cancer cell lines. RB1 mRNA expression is at normal levels for 5 of these 6 lines suggesting a greater importance of Brush-1 for breast cancer. Four primary breast tumors which showed no loss of heterozygosity in the 13q13-q14 region demonstrated normal levels of mRNA for both Brush-1 and RB1. However, four additional primary tumors which displayed loss of heterozygosity for this region had markedly decreased levels of Brush-1 mRNA while maintaining the normal levels for RB1. This differential loss of Brush-1 mRNA expression for both primary tumors and breast cancer cell lines is the expected pattern for a breast tumor suppressor gene.

Increased copy number at 20q13 in breast cancer: defining the critical region and exclusion of candidate genes.

Studies by comparative genomic hybridization have indicated that a major new locus for DNA amplification in breast cancer is 20q13 and suggested that this genetic event is associated with aggressive clinical behavior. We used interphase fluorescence in situ hybridization with anonymous cosmid probes and gene-specific P1 clones to determine the minimal common region of increased copy number and to study involvement of known genes at 20q13. Based on high-level copy number increases (3 to 10-fold) found with one or more probes in 5 of 14 (35%) breast cancer cell lines and in 3 of 36 (8%) primary tumors, the critical region was narrowed to approximately 1.5 megabases at 20q13.2 defined by fractional length pter values 0.81-0.84. Previously known genes were excluded as candidates, implying that this chromosomal region harbors a novel oncogene that contributes to the malignant progression of breast cancer.

The human homologue for the Caenorhabditis elegans cul-4 gene is amplified and overexpressed in primary breast cancers.

Amplification is a key mechanism whereby a cancer cell increases the message level of genes that confer a selective advantage when they are overexpressed. In breast cancer, there are many chromosome regions present in multiple copies relative to overall DNA copy number (amplicons), and their target genes are unknown. Using differential display, we have cloned and sequenced the full coding region of a candidate amplicon target gene located on chromosome 13. This candidate is the human homologue of the Caenorhabditis elegans cul-4 gene, cul-4A, a member of the novel cullin gene family, which is involved in cell cycle control of C. elegans. cul-4A was amplified and overexpressed in 3 of 14 breast cancer cell lines analyzed, and it was overexpressed in 8 additional cell lines in which it was not amplified. The latter observation, indicating that its overexpression can occur by mechanisms other than gene amplification, suggests that cul-4A plays a key role in carcinogenesis. Moreover, cul-4A was found to be amplified in 17 of 105 (16%) cases of untreated primary breast cancers, and 14 of 30 cases analyzed (47%) were shown by RNA in situ hybridization to overexpress cul-4A. These results suggest that up-regulation of cul-4A may play an important role in tumor progression.

ESX: a structurally unique Ets overexpressed early during human breast tumorigenesis.

The >30 known members of the Ets multigene family of transcriptional regulators are increasingly being recognized for their involvement in early embryonic development and late tissue maturation, directing stage-specific and tissue-restricted programs of target gene expression. Identifiable primarily by their 85 amino acid ETS DNA-binding domain and dispersed across all metazoan lineages into distinct subfamilies, Ets genes also produce malignancies in humans and other vertebrates when overexpressed or rearranged into chimeras retaining the ETS domain, suggesting that their oncogenic potential is determined by the program of target genes they regulate. Searching for Ets factors that regulate expression of the HER2/neu (c-erbB2) oncogene in human breast cancer, we identified a new epithelium-restricted Ets encoding an ETS domain homologous to the Drosophila E74/human Elf-1 subfamily, an amino-terminal region (A-region or Pointed domain) homologous to the distantly related Ets-1 subfamily, and a serine-rich box homologous to the transactivating domain of the lymphocyte-restricted High Mobility Group (HMG) protein, SOX4. Recombinant protein encoded by ESX (for epithelial-restricted with serine box) exhibits Ets-like DNA binding specificity in electrophoretic mobility shift assays and, in transient transfection assays, transactivates Ets-responsive promoter elements including that found in the HER2/neu oncogene. ESX is located at chromosome 1q32 in a region known to be amplified in 50% of early breast cancers, is heregulin-inducible and overexpressed in HER2/neu activated breast cancer cells. Tissue hybridization suggests that ESX becomes overexpressed at an early stage of human breast cancer development known as ductal carcinoma in situ (DCIS).

Regulation of insulin degradation: expression of an evolutionarily conserved insulin-degrading enzyme increases degradation via an intracellular pathway.

The insulin-degrading enzyme (IDE) is an evolutionarily conserved enzyme that has been implicated in cellular insulin degradation, but its site of action and importance in regulating insulin degradation have not been clearly established. We addressed this question by examining the effects of overexpressing IDE on insulin degradation in COS cells, using both human IDE (hIDE) and its Drosophila homolog (dIDE). The dIDE, which was recently cloned in our laboratory, has 46% amino acid identity with hIDE, degrades insulin with comparable efficiency, and is readily expressed in mammalian cells. Transient expression of dIDE or hIDE in COS monkey kidney cells led to a 5- to 7-fold increase in the rate of degradation of extracellular insulin, indicating that IDE can regulate cellular insulin degradation. Insulin-degrading activity in the medium was very low and could not account for the difference between transfected and control cells. To further localize the site of IDE action, the fate of insulin after receptor binding was examined. The dIDE-transfected cells displayed increased degradation of prebound insulin compared to control cells. This increase in degradation was observed even when excess unlabeled insulin was added to block reuptake or extracellular degradation. These results indicate that IDE acts at least in part within the cell. The lysosomotropic agents chloroquine and NH4Cl did not affect the increase in insulin degradation produced by transfection with dIDE, indicating that the lysosomal and IDE-mediated pathways of insulin degradation are independent. The results demonstrate that IDE can regulate the degradation of insulin by intact cells via an intracellular pathway.

Cloning and expression of the cDNA for a Drosophila insulin-degrading enzyme.

We have previously identified and characterized a metalloproteinase from Drosophila that cleaves insulin and transforming growth factor-alpha, but not epidermal growth factor, at physiological concentrations. On the basis of enzymatic properties and substrate specificity, this enzyme was identified as the Drosophila homolog of the mammalian insulin-degrading enzyme (IDE). We now report the cloning and sequencing of the cDNA coding for the Drosophila IDE (dIDE). Northern blot analysis indicates that the dIDE is translated from a 3.6-kilobase transcript similar in size to one of the two human IDE transcripts. The gene for the dIDE has been mapped to chromosome 3L (77B). The sequence of the dIDE is very similar to that of the human IDE, and both enzymes share limited but significant identity with the bacterial metalloproteinase protease III. Indirect studies based upon inhibitors, degradation products, and microinjected antibodies have suggested that the IDE can initiate cellular insulin degradation in mammalian cells. To determine whether dIDE expressed in mammalian cells can also degrade insulin, we transfected the cDNA into murine NIH3T3 cells. Extracts of the transfected cells showed increased insulin-degrading activity, demonstrating that the dIDE can be functionally expressed in mammalian cells. These results indicate that the properties of the IDE are evolutionarily conserved.

Insulin-degrading enzyme is differentially expressed and developmentally regulated in various rat tissues.

Insulin-degrading enzyme (IDE), a cytosolic metalloendoprotease, can degrade insulin, insulin-like growth factor-II, insulin-like growth factor-I, and transforming growth factor-alpha. While IDE has been implicated in the cellular degradation of insulin, other physiological functions of this enzyme are not known. To assess the possible role of IDE in cellular growth and development, we determined the tissue and developmental distribution of the enzyme. Rat IDE cDNA fragments and antibodies directed against human IDE were used to probe IDE transcripts and proteins in rat tissues. The results demonstrate that IDE transcripts are ubiquitous in rat tissues. The level of rIDE transcripts is high in adult rat testis, tongue, and brain; moderate in kidney, prostate, heart, muscle, liver, intestine, and skin; and low in spleen, lung, thymus, and uterus. The sizes of the major transcripts of rIDE are 3.4 and 6.3 kilobases in all tissues analyzed, except testis. Surprisingly, the highest level of rIDE mRNA in the adult rat was in the testis, and the major transcripts of rIDE in this tissue were shifted in size to 3.8 and 6.7 kilobases. Immunocytochemical analysis localized the rIDE mainly in the epithelium of prostate gland and kidney, and the cytosol of liver hepatocytes. During rat development from 6-7 days of age to adulthood, rIDE mRNA levels increased in brain, testis, and tongue; decreased in muscle and skin; and did not significantly change in other tissues examined. These studies reveal regulation of IDE or IDE-related genes in rat tissues and during rat development, suggesting that this enzyme may have multiple functions relating to cellular growth and development.

The mouse Id2 and Id4 genes: structural organization and chromosomal localization.

The Id proteins belong to a family of nuclear HLH proteins lacking a basic region and thought to function as dominant-negative regulators of bHLH proteins during cell growth and differentiation. In this paper, we report the genomic organization of the mouse Id2 and Id4 genes. These genes each span approximately 3 kb of the mouse genome and are each organized as three exons with recognizable splice donor and acceptor consensus sequences. Their genomic organization is very similar, consistent with their having evolved from a common, ancestral Id-like gene. Using FISH analysis, we have localized the mouse Id2 and Id4 genes to mouse chromosome 12 and 13, respectively.

Id gene expression during development and molecular cloning of the human Id-1 gene.

Id genes encode helix-loop-helix proteins that inhibit transcription by forming inactive heterodimers with basic helix-loop-helix (bHLH) proteins. bHLH proteins normally form either homodimers or heterodimers with other bHLH proteins and bind to a DNA sequence element activating transcription. Id-containing heterodimers are inactive because Id proteins lack the basic amino acid region necessary to form a DNA-binding domain. We have examined the relative levels of Id-1 and Id-2 mRNA during normal development and in malignant tissues. In the course of these experiments we cloned and sequenced the human Id-1 cDNA. Two related cDNA molecules encoding human Id-1 mRNAs were identified. Id-1a is a cDNA of 958 nucleotides and can encode a protein of 135 amino acids. Id-1b cDNA is 1145 nucleotides, can encode a protein of 149 amino acids, and appears to be a splice variant of Id-1a. The amino acid sequence of human Id-1 is greater than 90% homologous to that of mouse Id-1. The patterns of Id-1 and Id-2 expression during mouse development vary widely, and we detected Id-1 expression in human fetal and adult tissues from lung, liver, and brain. High Id-1 mRNA expression was found in many human tumor cell lines, including those isolated from nervous system tumors. We mapped Id-2 to human chromosome 2p25.