Chip is an essential cofactor for apterous in the regulation of axon guidance in Drosophila.

LIM-homeodomain transcription factors are expressed in subsets of neurons and are required for correct axon guidance and neurotransmitter identity. The LIM-homeodomain family member Apterous requires the LIM-binding protein Chip to execute patterned outgrowth of the Drosophila wing. To determine whether Chip is a general cofactor for diverse LIM-homeodomain functions in vivo, we studied its role in the embryonic nervous system. Loss-of-function Chip mutations cause defects in neurotransmitter production that mimic apterous and islet mutants. Chip is also required cell-autonomously by Apterous-expressing neurons for proper axon guidance, and requires both a homodimerization domain and a LIM interaction domain to function appropriately. Using a Chip/Apterous chimeric molecule lacking domains normally required for their interaction, we reconstituted the complex and rescued the axon guidance defects of apterous mutants, of Chip mutants and of embryos doubly mutant for both apterous and Chip. Our results indicate that Chip participates in a range of developmental programs controlled by LIM-homeodomain proteins and that a tetrameric complex comprising two Apterous molecules bridged by a Chip homodimer is the functional unit through which Apterous acts during neuronal differentiation.

Chip and apterous physically interact to form a functional complex during Drosophila development.

LIM homeodomain (LIM-HD) proteins play key roles in a variety of developmental processes throughout the animal kingdom. Here we show that the LIM-binding protein Chip acts as a cofactor for the Drosophila LIM-HD family member Apterous (Ap) in wing development. We define the domains of Chip required for LIM-HD binding and for homodimerization and show that mutant proteins deleted for these domains act in a dominant-negative fashion to disrupt Ap function. Our results support a model for multimeric complexes containing Chip and Ap in transcriptional regulation. This model is confirmed by the activity of a chimeric fusion between Chip and Ap that reconstitutes the complex and rescues the ap mutant phenotype.

Genetic background influences timing, morphology and dissemination of lymphomas in p53-deficient mice.

To examine the influence of genetic background on tumorigenesis in p53-deficient mice, we used selective breeding to produce congenic mice with a null p53 gene mutation introduced into the VM inbred strain. Cohorts of homozygous p53 null (-/-) mice from the original C57B6/129Sv mixed strain and the VM congenic strain were monitored for spontaneous tumor development, as were control cohorts of wild-type (+/+) and heterozygous (+/-) animals. Twenty-six of 28 C57B6/129Sv (-/-) mice died by the study end date (median survival =184.5 days). Twenty-three of 26 VM (-/-) mice died and their survival was significantly shorter (111 days, P<0.0001). Of 26 C57B6/129Sv (-/-) mice that died, 21 were autopsied: all 21 had lymphomas. Of 26 VM mice that died (23 -/-, 3 +/-), 21 were autopsied: 19 developed lymphoma and two had sarcomas. Several mice had additional neoplasms. Lymphomas in VM mice were distinct from those in C57B6/129Sv mice in that they i) arose on average more than two months earlier, ii) involved thymus more often than spleen or lymph nodes and iii) were more often poorly differentiated, high grade tumors. These results demonstrate that genetic background alone influences the onset, morphology and dissemination of lymphomas in p53-deficient mice and suggest the presence of genes which modify the timing and biological nature of lymphomas in these mice.

Gliomas in families: chromosomal analysis by comparative genomic hybridization.

Gliomas that aggregate in otherwise unremarkable families may have a heritable genetic basis. To determine the spectrum of genetic alterations in glioma-susceptible families, we examined tumor DNA from familial cases for regions of chromosomal gain or loss using comparative genomic hybridization (CGH). We compared chromosomal alterations within and among glioma families to those found in sporadic gliomas. A specific chromosomal abnormality common to the tumors of multiple unrelated probands with glioma or a specific chromosomal abnormality common to multiple affected persons in a single glioma-prone family would support the hypothesis of an inherited predisposition to glioma and at the same time identify specific regions of the genome harboring putative glioma susceptibility genes. Tumor DNA from 11 patients from seven families with two or more individuals with glioma was analyzed, including three members of a remarkable family having 10 affected individuals. We found no chromosomal abnormality common to all tumors of all probands nor did we find family-specific abnormalities in two of three glioma-prone kindreds. There were frequent copy number aberrations (CNAs) on chromosomes 7, 10, 19, and the sex chromosomes; other CNAs included +3q(13.3-29), -4q, +5q, -9q34, +12, -13q(21-->33), -15, -16p, +17qter, -18, -21, and -22. Amplifications occurred at +2 7p(11.1-->12), +2 7q(21.2-->33), +2 12q(13.2-->14), and +2 12p(11-->12). Although there were several novel CNAs [-16p, and +2 12p(11-p12)], none could readily explain the inheritance of these tumors.

Lack of germ-line mutations of CDK4, p16(INK4A), and p15(INK4B) in families with glioma.

Gliomas are tumors of the central nervous system that may be inherited in some patients. The gene(s) responsible for the clustering of gliomas in families have not yet been identified. Molecular studies of sporadic high-grade gliomas have revealed mutations or deletions of the genes encoding the protein kinase inhibitors p16(INK4A) and p15(INK4B) in a large proportion of tumors. Moreover, those tumors without deletions frequently display gene amplification and/or over-expression of mRNA encoding the protein kinase cdk4. We hypothesized that germ-line mutations in the p16(INK4A), p15(INK4B), or CDK4 genes might contribute to some cases of familial gliomas. To address this issue, we analyzed 36 kindreds with a predisposition to glial tumors. Genomic DNA from index members of these families was screened by PCR-single-strand conformational polymorphism analysis. We did not detect any functional mutations in the p16(INK4A), p15(INK4B), or CDK4 genes, although two individuals did have a previously described A140T polymorphism in p16(INK4A). Thus, despite the association between the sporadic forms of high-grade glioma and abnormalities of p16(INK4A), p15(INK4B), or CDK4, we found no evidence that germ-line mutations in the coding region of these three genes predispose to inherited glial tumors.

Loss of heterozygosity analysis of chromosomes 9, 10 and 17 in gliomas in families.

BACKGROUND: Studies of sporadic malignant gliomas have identified structural abnormalities in a number of chromosomal regions, especially losses of DNA on 9p, 10 and 17p. PURPOSE: We undertook the following molecular analysis in families with glioma to determine the frequency of chromosomal losses in these regions and to test the utility of microsatellite markers in demonstrating losses of heterozygosity. METHODS: Genomic DNA was extracted from tumor tissue and venous blood from 20 patients with a family history of glioma. Dinucleotide repeat polymorphisms (microsatellites) were analyzed by polymerase chain reaction to assess loss of constitutional heterozygosity (LOH) on 9p, 10 and 17p. Three polymorphic markers on chromosome 9 (D9S104, D9S161, D9S165), one on chromosome 10 (D10S209), and two on 17p (D17S786, D17S796) were used. Autoradiographic films were analyzed for LOH after radioactively labelled polymerase chain reaction products were resolved on denaturing formamide-acrylamide gels. RESULTS: Of 20 patients informative for at least one of three chromosome 9 markers, 12 (60%) showed LOH at one or more loci; of 9 informative for the chromosome 10 marker, 4 (44%) showed LOH; and of 16 informative for at least one of two chromosome 17 markers, 7 (44%) showed LOH at one or both loci. These LOH rates do not include instances of tumor nullizygosity (0-35%) and therefore represent minimum frequencies of chromosomal losses at these loci. CONCLUSIONS: Microsatellite markers can be used to detect LOH in archival glioma tissue. As in sporadic gliomas, frequent LOH was observed on 9p (9p21-22), 10 and 17p, supporting the notion that these regions may harbour tumor suppressor genes important in glioma development. Further work will be required to determine whether the proportion of LOH in these chromosomal regions is higher in familial gliomas than sporadic ones, as might occur with an inherited suppressor gene conferring susceptibility to gliomas in families.

p53 mutation, expression, and DNA ploidy in evolving gliomas: evidence for two pathways of progression.

BACKGROUND: Two lines of evidence indirectly implicate the tumor suppressor p53 (also known as TP53) gene in glioma development. First, germline mutations of the p53 gene are associated with increased susceptibility to glioma. Second, chromosome 17p deletions and p53 gene mutations are found frequently in sporadic gliomas of all malignancy stages. These observations suggest that mutations of the p53 gene may be early events in glioma development. PURPOSE: Our purpose was to analyze 15 low-grade astrocytic gliomas that progressed to higher-grade gliomas, examining the status of the p53 gene in both the initial and recurrent tumors. Also, we explored the relationships between p53 status, DNA ploidy, tumor grade, and patient survival. METHODS: Fifteen low-grade gliomas that recurred as tumors of higher grade 17-102 months after initial treatment (biopsy, resection, radiotherapy, or chemotherapy) were identified from hospital records of patients (eight male and seven female) aged 31-68 years. Pathologic diagnosis was re-evaluated. Polymerase chain reaction (PCR)-single-strand conformation polymorphism and DNA sequencing were performed on tissue samples from the initial and recurrent tumors of each patient, using oligonucleotide PCR primers directed to exons 5-9 of the p53 gene. p53 expression was determined by immunohistochemistry and DNA ploidy evaluated by DNA flow cytometry. RESULTS: Eight (53%) of fifteen tumors had p53 mutations in exons 5-9. Nine (64%) of fourteen were immunopositive initially, and eight of these were also immunopositive at recurrence. p53 gene status was significantly associated with p53 expression in the initial tumor (P = .02), and p53 expression at initial diagnosis was significantly related to tumor pathology at recurrence (P = .03). Patients with p53 mutant tumors survived nearly twice as long as those without mutations (median survival, 61 versus 33 months; P = .031). There was no significant difference in recurrence-free survival between patients with p53 mutant and nonmutant tumors (48 versus 33 months; P = .37), but there was a significant difference in postrecurrence survival (17 versus 2 months; P = .019). CONCLUSION: Low-grade tumors that recurred as anaplastic gliomas were characterized by p53 gene mutation, immunopositivity, and DNA non-diploidy. Low-grade tumors that recurred as glioblastomas generally had intact p53 genes and were immunonegative. These findings suggest that histologically indistinguishable, low-grade astrocytic gliomas that are destined to progress to higher grades, do so along two distinct clinicopathologic pathways (either stepwise to anaplastic glioma, then glioblastoma, or directly to glioblastoma) marked by the presence or absence of p53 mutation.

p53 and ras gene expression in human esophageal cancer and Barrett's epithelium: a prospective study.

To assess potential clinical applications for molecular genetic markers associated with human esophageal tumorigenesis, ten patients with primary esophageal adenocarcinomas were studied prospectively to evaluate expression of the p53 and H-ras genes. Total RNA was extracted from tumor, Barrett's epithelium, and histologically normal esophageal mucosa obtained at surgical resection, and gene expression investigated by Northern blot analysis. p53 was overexpressed, relative to normal tissue from the same patient, in seven tumor and six Barrett's specimens, whereas high levels of H-ras were found in only four tumor and one Barrett's specimen. Clinical correlative data were obtained for all patients, with a median follow-up of 14 months. Advanced pathologic stage was associated with poor survival. No association was found between gene expression and outcome. Three patients with low p53 and H-ras levels developed metastatic disease 7 to 12 months following resection. We conclude that both p53 and ras are implicated in the progression of Barrett's epithelium to invasive cancer, and that further clinical correlative studies are warranted to evaluate potential clinical application for such molecular markers.

Absence of hereditary mutations in exons 5 through 9 of the p53 gene and exon 24 of the neurofibromin gene in families with glioma.

Inherited mutations of the p53 and neurofibromin genes are thought to cause two distinct neoplastic disorders in which gliomas occur, the Li-Fraumeni syndrome and neurofibromatosis type 1. We investigated the possibility that inherited mutations in specific regions of these genes also contributed to the clustering of gliomas in otherwise normal families. Twenty-six members of 16 families with glioma were screened for germline mutations of exons 5 through 9 of the p53 gene and exon 24 of the neurofibromin gene using a polymerase chain reaction-single-strand conformation polymorphism method. No germline mutations were found, suggesting that the genetic basis of familial glioma is distinct from that of gliomas occurring in the Li-Fraumeni syndrome, and that inherited mutations of the catalytic domain of neurofibromin do not predispose affected glioma families to these tumors.

Gliomas in families.

This is a descriptive study of 19 families with glial tumors. Twelve were identified prospectively from 178 consecutive, unrelated adults and children with newly diagnosed gliomas seen at a regional cancer center between 01 Jan 89 and 31 Mar 91 (6.7%). There were 45 affected members (42 confirmed); 30 males, 15 females, ages 4 months-78 years (median, 44.5 years; mean, 38.9 years). Two families had four affected members, three families had three, and the others two. All confirmed tumors were supratentorial and all, save one, contained an astrocytic element. Three additional members of two families had other brain or neuroectodermal tumors. These families were not unusually cancer prone and did not appear to have neurofibromatosis, tuberous sclerosis, or colonic polyposis. There was no consistent pattern of inheritance.