Effects of the MYC oncogene antagonist, MAD, on proliferation, cell cycling and the malignant phenotype of human brain tumour cells.

To investigate how overexpression of MAD, an antagonist of MYC oncogenes influences the malignant phenotype of human cancer cells, an adenovirus vector system was used to transfer the human MAD gene (AdMAD) into human astrocytoma cells. Decreased growth potential of AdMAD-infected cells was evidenced by a decrease in [3H]thymidine incorporation, an increase in cell doubling time and alteration of cell-cycle distribution. Diminished malignant potential of AdMAD-infected cells was manifested by their loss of anchorage-independent growth in soft agar and by their inability, in general, to induce tumorigenesis in a xenograft animal model. These studies indicate that adenovirus constructs encoding MAD dramatically inhibit the proliferation and tumorigenicity of human astrocytoma cells and support the use of MAD for gene therapy of human tumours.

Nonrandom X chromosome DNA methylation patterns in hemophiliac females.

Molecular X chromosome inactivation analysis was used to characterize three females (and their families) with severe hemophilia. First, the maternal and paternal X chromosomes were distinguished by restriction fragment length polymorphisms (RFLPs). Second, the patterns of methylation of X chromosome genes using methylation-sensitive restriction endonucleases were determined. Of the six X chromosome probes tested, only the phosphoglycerol-kinase (PGK) and hypoxanthine-phosphoribosyl-transferase (HPRT) clones were informative, indicating that other X chromosome probes are not useful for X inactivation analysis. After digestion with Hpa II or Hha I, the hybridization intensity of the RFLPs of all three mothers and an unaffected sister were diminished by 50%, consistent with random X chromosome inactivation. The methylation patterns of the X chromosomes of the affected females, however, were clearly nonrandom. Depending upon the probe and the patient, HPRT and PGK sequences were either completely methylated or unmethylated. These findings are extremely suggestive that nonrandom X chromosome inactivation (lyonization) is the basis for severe hemophilia in these females.

Tumor suppression and inhibition of aneuploid cell accumulation in human brain tumor cells by ectopic overexpression of the cyclin-dependent kinase inhibitor p27KIP1.

To investigate how overexpression of p27KIP1, a downstream effector of TGF-beta and a universal cyclin-dependent kinase (CDK) inhibitor could influence the malignant phenotype of malignant human brain tumor cells, an adenovirus vector system was used to transfer the human p27KIP1 gene (Adp27KIP1) into the human astrocytoma cell line, U-373MG. Inhibition of CDK activity in Adp27KIP1-infected cells was indicated by inhibition of [3H]thymidine incorporation, an increase in cell doubling time and by cell cycle arrest in G1. Notably, ectopic overexpression of p27KIP1 was associated with a marked decrease in the accumulation of aneuploid cells. Diminished malignant potential of Adp27KIP1-infected cells was manifested by the loss of anchorage-independent growth in soft agar and by the inability to induce tumorgenesis in a xenograft model. These studies suggest that p27KIP1 is a tumor suppressor gene and supports the use of Adp27KIP1 for gene therapy of human brain tumors.

Downregulation of cyclin-dependent kinase 2 activity and cyclin A promoter activity in vascular smooth muscle cells by p27(KIP1), an inhibitor of neointima formation in the rat carotid artery.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) contributes to intimal hyperplasia during atherosclerosis and restenosis, but the endogenous cell cycle regulatory factors underlying VSMC growth in response to arterial injury are not well understood. In the present study, we report that downregulation of cyclin-dependent kinase 2 (cdk2) activity in serum-deprived VSMCs was associated with the formation of complexes between cdk2 and its inhibitory protein p27(KIP1) (p27). Ectopic overexpression of p27 in serum-stimulated VSMCs resulted in the inhibition of cdk2 activity and repression of cyclin A promoter activity. Collectively, these findings indicate that p27 may contribute to VSMC growth arrest in vitro. Using the rat carotid model of balloon angioplasty, a marked upregulation of p27 was observed in injured arteries. High levels of p27 expression in the media and neointima correlated with downregulation of cdk2 activity at 2 wk after angioplasty, and adenovirus-mediated overexpression of p27 in balloon-injured arteries attenuated neointimal lesion formation. Thus, the inhibition of cdk2 function and repression of cyclin A gene transcription through the induction of the endogenous p27 protein provides a mechanism for the inhibition of VSMC growth at late time points after angioplasty.

Temperature-dependent modulation of lipopolysaccharide-induced interleukin-1 beta and tumor necrosis factor alpha expression in cultured human astroglial cells by dexamethasone and indomethacin.

In bacterial meningitis, LPS induces production in cerebrospinal fluid of the cytokines IL-1 beta and tumor necrosis factor alpha (TNF alpha), which are the principle mediators of meningeal inflammation. IL-1 beta and TNF alpha induce fever, and elevated temperature may affect cytokine expression. Dexamethasone treatment improves outcome in bacterial meningitis possibly by inhibiting IL-1 beta and TNF alpha. In this report, the effects of elevated temperature and dexamethasone on LPS-stimulated IL-1 beta and TNF alpha mRNA gene expression and protein synthesis were studied in human astrocytoma cell lines and primary cultures of human fetal astrocytes. Cells cultured at 40 degrees C exhibited smaller peaks of IL-1 beta and TNF alpha transcription and protein synthesis compared with cells cultured at 37 degrees C. The addition of dexamethasone before, during, or after exposure of the cells to LPS resulted in temperature-dependent inhibition of IL-1 beta transcription and protein synthesis. The most extensive inhibition occurred in pretreated cells cultured at 37 degrees C. Cotreatment with LPS and dexamethasone also inhibited TNF alpha mRNA transcription at both temperatures. The effects of another antiinflammatory agent, indomethacin, on LPS induction of IL-1 beta and TNF alpha mRNA were temperature and cell line dependent. These findings provide a possible explanation for the efficacy of dexamethasone treatment of bacterial meningitis and support the proposal that fever may be beneficial to the host in this disease.

Nonrandom distribution of N-myc oncogene genotypes in neuroblastoma.

The distributions of Pvu II and Sph I alleles of the N-myc oncogene (also known as MYCN) were studied in a series of normal individuals and pediatric patients with solid tumors. In the case of Pvu II, where the polymorphic site is located 3' of the gene, the frequencies of the allele were 0.27 (11-kilobase fragment) and 0.73 (8-kilobase fragment) in 43 unrelated normal Caucasians. The frequencies of the allele were similar in 40 non-N-myc-amplified neuroblastomas, 47 Wilms' tumors, and 31 other pediatric tumors. In these cases, the genotypes were in Hardy-Weinberg equilibrium. In 18 N-myc-amplified neuroblastomas, however, the observed genotype frequencies deviated from Hardy-Weinberg equilibrium (P less than .005). Similar observations were made with an Sph I restriction fragment length polymorphism where the polymorphic site is located in intron 2. The differences between amplified and nonamplified neuroblastomas suggest a possible involvement of sequences at or near N-myc in the progression of tumors where the N-myc gene is amplified.

N-myc oncogene RNA expression in neuroblastoma.

Tumor specimens from 33 patients with neuroblastoma were assayed for amplification of the N-myc oncogene and RNA expression to determine whether N-myc RNA expression levels correlated with N-myc gene amplification and clinical outcome. N-myc gene amplification was detected in one stage II tumor, one stage IV-S tumor, and seven stage III or IV tumors. In each case, N-myc RNA expression roughly paralleled N-myc gene amplification. However, enhanced N-myc RNA expression was not confined to tumors with N-myc gene amplification: all of the early (stage I and II) tumors, five stage IV-S tumors, and 12 advanced (stage III and IV) tumors had levels of N-myc RNA that were elevated up to 50-fold. While N-myc gene amplification correlated with prognosis, there was no such correlation with levels of N-myc RNA expression. The precise role of the N-myc gene in the pathogenesis of neuroblastoma remains unclear.

Allelic loss at chromosomes 3p, 8p, 13q, and 17p associated with poor prognosis in head and neck cancer.

BACKGROUND: Little is known about the molecular genetic events that contribute to the pathogenesis of squamous cell carcinoma of the upper aerodigestive tract. Previous molecular genetic studies have been limited to the identification of mutations of the p53 (also known as TP53) tumor suppressor gene, activation of a limited set of oncogenes, allelic loss at 3p and other locations, and occasional association with human papillomavirus infection. PURPOSE: Our purpose was to screen tumor tissue and blood from patients with squamous cell carcinoma of the upper aerodigestive tract for loss of heterozygosity at polymorphic loci corresponding to each of the autosomal chromosomes and to identify the locations of additional putative tumor suppressor genes, other than RB (also known as RB1) and p53, that may contribute to the pathogenesis of this disease. METHODS: Tumor tissue and blood were obtained from 68 consecutive patients with squamous cell carcinoma of the upper aerodigestive tract. In all cases, tumor tissue was obtained from the center of the surgical specimen. The relative absence of non-neoplastic tissue was confirmed by frozen-section histologic examination of immediately adjacent tissue. Initially, 30 paired tissue and blood samples were tested for loss of heterozygosity by polymerase chain reaction (PCR) to amplify 43 different highly polymorphic sequences containing small oligonucleotide repeats. After PCR amplification, with unique oligonucleotides flanking the repeat, visualization and sizing of the alleles on DNA sequencing gels were performed. Specific loss of heterozygosity was distinguished from random genetic loss due to generalized chromosomal instability if it occurred in more than 20% of specimens tested for a particular marker. RESULTS: Significant loss of heterozygosity (> 20%) occurred at alleles at chromosome bands 3p21 (32%), 3p25-26 (56%), 8pter-21.1 (31%), 13q14 (27%), and 17p12 (45%). Loss of heterozygosity at more than two loci was significant with a poor prognosis (P = .039). CONCLUSIONS: These findings demonstrate that squamous cell carcinoma of the upper aerodigestive tract exhibits genetic alterations at multiple loci and that allelic loss at more than two locations is indicative of a poor prognosis (the likelihood of the patient dying of disease). IMPLICATIONS: While tumor suppressor genes at 3p (VHL), 13q (RB), and 17p (p53) have been identified, altered genes at other loci on 3p and on 8p have not yet been characterized. Furthermore, the genotype at these loci for squamous cell carcinoma of the upper aerodigestive tract has prognostic importance and may identify the patients who should receive the most aggressive treatment.

Infrequency of MDM2 gene amplification in pediatric solid tumors and lack of association with p53 mutations in adult squamous cell carcinomas.

Loss of function of the p53 tumor suppressor gene by point mutation is the most commonly detected genetic alteration in human cancer. There is growing evidence that amplification and overexpression of the MDM2 gene are alternative mechanisms that also lead to functional inactivation of p53. While p53 mutations and MDM2 amplification have been reported to occur in rhabdomyosarcoma and osteogenic sarcoma, the incidence of MDM2 in other pediatric solid tumors is not known. We therefore tested a series of other pediatric solid tumors for MDM2 gene amplification. MDM2 amplification could not be detected in specimens from 40 Wilms' tumors, 15 neuroblastomas, 12 sarcomas, or 4 hepatoblastomas tested. To determine whether MDM2 amplification was an alternative mechanism of p53 inactivation in adult carcinomas that frequently possess p53 mutations, 68 samples of squamous cell carcinomas of the upper aerodigestive tract, 24% of which were previously shown to contain p53 mutations, were also tested for MDM2 amplification. MDM2 amplification did not occur in any of the tumor specimens tested. These findings suggest that MDM2 amplification may only occur in a limited subset of human tumors. Loss of function of p53 may be an essential event in human tumorigenesis. If so, then other mechanisms of p53 inactivation must occur in those tumors that exhibit neither p53 mutation nor MDM2 amplification.

Enhanced expression of the N-myc gene in Wilms' tumors.

Activation of myc-family oncogenes has been implicated in the genesis of a variety of neoplasms. In addition, these genes exhibit specific patterns of expression during murine development. We now report that N- and c-myc are differentially expressed in normal developing human renal tissues and in Wilms' tumor, a neoplasm which derives from primitive kidney cells. Twelve of 13 Wilms' tumors tested exhibited greatly enhanced levels of expression which occurred in the absence of gene amplification. We also detected N-myc expression in other primitive neoplasms including medulloblastoma and hepatoblastoma. Our observations suggest that N-myc expression is not limited to neuroectodermal tumors as was previously thought, but is a marker for several neoplasms that derive from primitive cell precursors. Finally, high level expression of N-myc was associated with markedly diminished levels of c-myc, suggesting that enhanced expression of N-myc gene might lead to down-regulation of c-myc.

A glial-specific, repressible, adenovirus vector for brain tumor gene therapy.

The principle hurdles for gene therapy are selectivity and efficacy. Toward that end, we constructed an adenovirus gene delivery system to enable robust, glial-specific, and repressible ectopic expression. A replication-incompetent (E1-deleted) adenovirus 5 vector was modified by the addition of three tandem repeats of a 300-bp fragment enhancer region of the glial fibrillary acidic protein gene coupled to a minimal promoter sequence from human cytomegalovirus to drive a tetracycline-controlled transactivator. Using beta-galactosidase as a reporter gene, we demonstrated high level expression in cells of glial origin (including cell lines derived from glioblastoma multiforme) but no detectable expression in nonglial cells (neuroblastoma or fibroblasts). Furthermore, expression was tightly regulated by anhydrous tetracycline. To our knowledge, this is the first gene therapy delivery system that is glial specific and which also allows for repression of ectopic gene expression.

Frequency and diversity of p53 mutations in childhood rhabdomyosarcoma.

The p53 gene was examined in primary or metastatic tumors from six patients with rhabdomyosarcoma (RMS) and in five RMS cell lines by screening methods including single-strand conformation polymorphism analysis, the RNase protection assay, sequencing of complementary DNA subclones, and Southern blotting. Six original tumors were of embryonal histology, four alveolar, and one mixed. p53 mutations were identified in four of the six tumors or cell lines derived from tumors with embryonal histology and in one of the four with alveolar histology. Consistent with p53 allele loss, each mutation was found in the homo- or hemizygous state. One tumor showed a G to C transversion at p53 codon 213 (arginine to proline), and another showed deletion of the entire gene. The p53 mutations in cell lines included a codon 248 C to T transition (arginine to tryptophan) in RD and a codon 280 A to T transversion (arginine to serine) in RH30. The cell line CTR contained a 4-base pair deletion at codons 219/220 in exon 6 with resultant frame shift and premature termination in exon 7. These data support the role of diverse types of p53 mutations in the pathogenesis and/or progression of a significant proportion of cases of childhood RMS.

Genetic alterations of chromosome band 9p21-22 in head and neck cancer are not restricted to p16INK4a.

Although genetic alterations of chromosome band 9p21-22 occur frequently in head and neck squamous cell carcinoma (HNSCC) cell lines, alterations of the cyclin-dependent kinase inhibitor p16INK4a located in this region are less common in corresponding primary tumors. To further investigate genetic alterations at 9p21-22 and p16INK4a in primary HNSCC, a paired set of 21 tumors and blood specimens that were shown previously to exhibit allelic loss at 3p and elsewhere, were tested for LOH at 9p21-22 using eight different highly polymorphic marker. Sixteen of the samples (81%) exhibited LOH for at least one marker. Frequent LOH was found surrounding p16INK4a and at three additional non-contiguous regions of 9p21-22. No homozygous deletions were identified. SSCP screening and direct sequence analysis led to the identification of mutations the p16INK4a gene in two tumors. p16INK4a was not hypermethylated in any of the samples studied. Furthermore, there was no correlation between LOH at 9p21-22 and the RB1 tumor suppressor gene. These findings indicate that in the set of tumors that we tested, LOH at 9p21-22 is common in primary HNSCC but that genetic alterations of p16INK4a located in this region are unusual. Additional tumor suppressor genes at 9p21-22 may therefore be involved in the pathogenesis of this tumor.

Frequent allelic loss at chromosome arm 3p is distinct from genetic alterations of the Von-Hippel Lindau tumor suppressor gene in head and neck cancer.

Previous molecular genetic studies revealed that allelic loss of chromosome arm 3p is a frequent event in upper aerodigestive tract squamous cell carcinoma (UADT SCC). Recently, the Von-Hippel Lindau (VHL) tumor suppressor gene was identified at chromosome band 3p25-26. To determine if the VHL locus is altered in these tumors, a paired series of 26 tumors and blood from patients with UADT SCC that were previously shown to exhibit allelic loss of 3p were tested for LOH surrounding the VHL locus using four different polymorphic markers. All of the samples (100%) exhibited LOH for at least 1 marker. However, no LOH was detected using a polymorphism within exon 1 of the VHL gene which was informative for 18 of the 26 cases. Furthermore, mutations of the VHL gene could not be identified by single-strand conformation polymorphism, dideoxyfingerprint or direct DNA sequence analysis. In addition, the VHL gene was not inactivated by hypermethylation in any of the 26 tumor samples studied. These findings demonstrate that allelic loss of chromosome arm 3p in UADT SCC involves regions surrounding the VHL locus but does not include the VHL gene. The VHL gene, therefore, does not appear to be involved in the pathogenesis of UADT SCC.

Effects of ectopic overexpression of p21(WAF1/CIP1) on aneuploidy and the malignant phenotype of human brain tumor cells.

p21WAF1/CIP1 is a downstream effector of the p53 tumor suppressor gene and a universal cyclin-dependent kinase (CDK) inhibitor. To determine the ability of p21WAF1/CIP1 to function as a tumor suppressor, we constructed a replication-defective adenovirus vector containing p21WAF1/CIP1 (Adp21WAF1/CIP1) to effect ectopic overexpression in a p53-defective human astrocytoma cell line, U-373MG. We observed a marked decrease in CDC2 and CDK2 kinase activity associated with a corresponding decrease in the amount of CDC2 but not CDK2 protein; a decreased growth potential of Adp21WAF1/CIP1-infected cells demonstrated by diminished [3H]thymidine incorporation, increased cell doubling time and G1-arrested cell cycle; an association between Adp21WAF1/CIP1-infected cells and inhibition of aneuploid cell accumulation; and an alteration of the malignant phenotype of cells was evidenced by the loss of anchorage-independent growth in soft agar and the failure to induce tumorigenesis in both peripheral and intracerebral xenograft models, including the prevention of tumor formation Adp21WAF1/CIP1 infection 2 days post tumor cell implantation. Adp21WAF1/CIP1. Adp21WAF1/CIP1 appears to be a strong candidate for gene therapy studies based on these studies indicating that Adp21WAF1/CIP1 inhibits proliferation, tumorigenicity and aneuploidy in human brain tumor cells.

Cytogenetics of a renal cell carcinoma in a 17-month-old child. Evidence for Xp11.2 as a recurring breakpoint.

A renal cell carcinoma from a 17-month-old boy with a history of maternal hydrocarbon exposure was found to have a 46,Y,t(X;17)(p11.2;q25) karyotype. Although this translocation has not previously been reported, other translocations involving Xp11.2 have been described, suggesting that this may represent a non-random breakpoint involved in the pathogenesis of childhood renal cell carcinoma. Both chromosomes 3 in the tumor were normal by both karyotype and RFLP analysis.

p53, retinoblastoma, and human papillomavirus in squamous cell carcinoma and adjacent normal mucosa of the upper aerodigestive tract.

OBJECTIVE: The primary objective of this study was to determine the incidence of p53 and retinoblastoma tumor suppressor gene mutations and human papillomavirus infection in squamous cell carcinoma and adjacent normal mucosa of the upper aerodigestive tract. The secondary objective was to associate these findings with clinical and histopathologic features. DESIGN: Point mutations of p53 were identified by single-strand conformation polymorphism analysis and confirmed by direct DNA sequence analysis. Polymerase chain reaction-based methods were used to identify loss of heterozygosity of the retinoblastoma tumor suppressor gene and the presence of human papillomavirus sequences. SETTING: University-based tertiary care center. PATIENTS OR OTHER PARTICIPANTS: Forty-five consecutive cases of upper aerodigestive tract squamous cell carcinoma. RESULTS: Eleven point mutations of p53 were identified in tumor samples (24%). No functional p53 mutations were detected in adjacent normal tissue from eight of these individuals nor was there evidence of p53 alteration in normal tissue adjacent to 12 of 30 additional tumors tested that demonstrated conformational alterations by single-strand conformation polymorphism analysis. The p53 mutations were significantly associated with local invasion. Loss of heterozygosity (which has a 20% chance of random occurrence in tumors) was detected at the retinoblastoma locus in 15% of the tumors tested. Five of the specimens (11%) were positive for human papillomavirus sequences (two of which also contained p53 mutations). CONCLUSIONS: These findings suggest that p53 but not retinoblastoma or human papillomavirus is an important prognostic factor and is involved as a late event in the pathogenesis of upper aerodigestive tract squamous cell carcinoma.

Inhibition of lipopolysaccharide-induced IL-1 beta transcription by cyclic adenosine monophosphate in human astrocytic cells.

The response to LPS includes synthesis by monocytes of the inflammatory mediator IL-1 beta. Although the intracellular signaling pathways activated by LPS that lead to IL-1 beta production have been studied extensively in monocytes, these pathways have not been investigated in astrocytes, an important source of IL-1 beta in the central nervous system. cAMP has been implicated in LPS signaling as a positive regulator of IL-1 beta mRNA accumulation in monocytes. In this study, we demonstrate that in human astrocytes (both fetal and the astrocytoma cell line, U-373 MG), agents that elevate intracellular cAMP decrease LPS-induced IL-1 beta mRNA accumulation. Elevated intracellular cAMP does not affect IL-1 beta mRNA stability, but inhibits LPS-induced transcription initiation of IL-1 beta in U-373 MG cells. Elevated intracellular cAMP may be a negative feedback regulatory mechanism to inhibit IL-1 beta production employed by astrocytes that (unlike monocytic cells) lack a glycosyl-phosphatidylinositol (GPI)-anchored form of the LPS receptor, CD14. Whether cAMP inhibits an LPS-inducible signaling pathway or negatively affects cAMP-dependent transcription factors remains to be determined.

Random X chromosome inactivation in a female with a variant of Wiskott-Aldrich syndrome.

A 15-month-old female presented with eczema, thrombocytopenia, recurrent infections and failure to thrive. She had low serum IgM and IgG subclasses and an abnormal lymphocyte proliferative response to periodate in vitro. Molecular X chromosome inactivation analysis, using the polymorphic HUMARA DNA probe, showed that the infant has random X chromosome inactivation. We conclude that she has an atypical form of Wiskott-Aldrich syndrome which may be inherited in an autosomal recessive manner.