Overexpression of Mad transcription factor inhibits proliferation of cultured human hepatocellular carcinoma cells along with tumor formation in immunodeficient animals.

BACKGROUND: Dominant negative regulation of critical cell cycle molecules could perturb survival of cancer cells and help develop novel therapies. METHODS: To perturb the activity of c-Myc, which regulates G0/G1 transitions, we overexpressed Mad1 protein with an adenoviral vector, AdMad. Studies were conducted with established cell lines, including HepG2, HuH-7 and PLC/PRF/5 liver cancer cells, RAT-1A embryonic fibroblasts and U373MG astrocytoma cells. RESULTS: After AdMad-treatment, transduced cells exhibited decreased proliferation rates in culture conditions. RAT-1A embryonic fibroblasts and U373MG astrocytoma cells showed accumulations in G0/G1, whereas HepG2 and HuH-7 cells accumulated in G0/G1, and additionally in G2/M, albeit to a lesser extent. An in vitro assay using hepatocyte growth factor to stimulate proliferation in HuH-7 cells showed blunting of growth factor responsiveness, along with inhibition of cell cycle progression in AdMad-treated cells. No cytotoxicity was observed in AdMad-treated cells in culture, although cells lost clonogenic capacity in soft agar. In vivo assays using HepG2 cell tumors in immunodeficient mice showed that overexpression of AdMad prevented tumorigenesis. CONCLUSIONS: These studies indicate roles of Mad in G2/M, as well as the potential of manipulating cell cycle controls for treating liver cancer.

Taxol, vincristine or nocodazole induces lethality in G1-checkpoint-defective human astrocytoma U373MG cells by triggering hyperploid progression.

In this report, we describe a novel lytic mechanism exploited by antimicrotubule drugs (AMDs) such as Taxol which are frequently used to treat multiple human cancers including breast and ovarian cancers. In cells lacking the G1-arresting capacity due to the defect in retinoblastoma or p53 gene function, AMDs trigger hyperploid progression and death. The hyperploid progression occurs via continued cell-cycle progression without cell division. Blocking hyperploid progression through hydroxyurea or ectopically expressed p27(Kip1), a G1-specific Cdk inhibitor, abrogates AMD cytotoxicity. Thus, AMDs induce lethality in G1-checkpoint-defective cells by triggering hyperploid progression. The phenomenon is reminiscent of that observed previously with bub-1 yeast mutant. The potential significance of this finding lies in that hyperploid progression-mediated death may be exploited to develop a therapy with tumor-specificity at the genetic level. As a large fraction of human cancers are mutated in p53 gene, it may have a wide therapeutic applicability.

Multipoint analysis of human chromosome 11p15/mouse distal chromosome 7: inclusion of H19/IGF2 in the minimal WT2 region, gene specificity of H19 silencing in Wilms' tumorigenesis and methylation hyper-dependence of H19 imprinting.

WT2 is defined by maternal-specific loss of heterozygosity (LOH) on chromosome 11p15.5 in Wilms' tumors (WTs). The imprinted H19 gene, in this region, is silenced and hypermethylated in most WTs, and this is linked to pathological biallelic expression of IGF2. However, H19 and IGF2 lie within a larger imprinted domain, and the gene specificity of H19 epimutation has been a persistent question. To address this, we assessed LOH, gene expression and DNA methylation at multiple sites in and around the imprinted domain. LOH mapping showed that the entire domain, including IGF2/H19, is within the minimal WT2 region. Genes within the domain, including IPL/TSSC3/BWR1C, IMPT1/ORCTL2/BWR1A/TSSC5, KvLQT1/KCNA9 and TAPA1/CD81, as well as the zinc finger gene ZNF195/ZNFP104 near the centromeric border, were expressed persistently in many WTs. DNA hypermethylation was not detected with 5" upstream probes for IPL, IMPT1, KvLQT1 and ZNF195 in WTs or WT-associated kidneys. Fully developed WTs showed variable hypomethylation at an imprinted CpG island in a KvLQT1 intron, but this was only complete in the cases with LOH and was not observed in pre-neoplastic WT-associated kidneys with H19 epimutation. Analysis of the corresponding region of mouse chromosome 7 using methyltransferase-hypomorphic mice showed that the H19 imprint was fully erased, but that the allelic bias at Ipl, Impt1, p57 Kip2 and, to a lesser extent, Kvlqt1, persisted. Pre-existing massive allelic asymmetry for DNA methylation and hyper-dependence of transcription on methylation status may underlie the mechanism of gene-specific silencing of H19 in Wilms' tumorigenesis.

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.

The use of early postoperative MR in detecting residual juvenile cerebellar pilocytic astrocytoma.

PURPOSE: We compared visibility of residual juvenile cerebellar pilocytic astrocytomas (JPAs) on early postoperative and follow-up MR studies to determine whether early postoperative MR imaging has a valid role as a baseline study. METHODS: We reviewed the MR images of 21 consecutive children who had undergone resection of cerebellar JPA. The diagnosis of residual tumor was made on the basis of nodular enhancement that corresponded to enhancing tumor on the preoperative MR studies and/or nonenhancing nodular T2 signal that corresponded to nonenhancing tumor. Because no patient received chemotherapy or radiation therapy, abnormal T2 signal or enhancement on the early postoperative study that resolved on the follow-up study was presumed to be due to peritumoral edema and/or surgical manipulation. Nodular T2 signal and/or enhancement in the tumor bed not seen on the initial postoperative MR study but present on the subsequent MR study and unchanged on serial follow-up MR studies was presumed to represent residual tumor rather than tumor that had recurred. RESULTS: Compared with follow-up studies, the initial postoperative MR images were true-positive for residual tumor in six patients, false-positive in five, equivocal for residual tumor in four, true-negative in five, and false-negative in one. Residual tumor did not consistently enhance, and peritumoral edema and changes resulting from surgical manipulation tended to mask or simulate residual tumor. CONCLUSION: Early postoperative MR imaging is not accurate in differentiating residual JPA from postoperative changes, and the role of early postoperative MR imaging as a baseline study for comparison with further studies is questionable.

Infrequency of BRCA2 alterations in head and neck squamous cell carcinoma.

Alterations of BRCA2 result in increased susceptibility to breast cancer in both men and women (relative lifetime risks of 0.06 and 0.8 respectively). BRCA2 maps to 13q12-q13 and encodes a transcript of 10,157 bp. Other cancers that have been described in BRCA2 mutation carriers include those of the larynx. Human chromosome 13q has been shown previously by LOH studies to harbor several tumor suppressor genes for head and neck squamous cell carcinoma (HNSCCs). We therefore examined the role of BRCA2 in the development of these cancers. Only 6/22 (27%) of the laryngeal cancers we examined demonstrated LOH of the BRCA2-containing region. These and 10 other HNSCCs of different origins that were demonstrated by LOH studies to have lost the region of chromosome 13 containing BRCA2 were examined for alterations in this gene. SSCP analysis failed to reveal any alterations leading us to conclude that BRCA2 alterations are not frequently involved in the pathogenesis of HNSCCs and that the observed LOH of chromosome 13 loci is due to other, as yet, unidentified tumor suppressor gene(s). Interestingly tumors with LOH in this region (proximal to D13S118) were far more likely to be derived from women than men. This is unusual since HNSCCs are usually fourfold more common in men than in women.

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.

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.

Genomic imprinting and Wilms' tumor.

The selective loss of maternal and reduplication of paternal chromosome 11p15.5 alleles in Wilms' tumors (WTs) points to the existence of a paternally imprinted tumor suppressor gene(s) and/or a maternally imprinted dose-dependent growth-promoting gene(s) in this chromosomal region. Two reciprocally imprinted chromosome 11p15.5 genes, H19, a candidate tumor suppressor gene, and IGF2, a candidate dominant oncogene, have been well-characterized in terms of their imprinting and expression status in WTs. Here we review and extend data indicating that a majority of WTs show a bipaternal epigenotype at these loci, with H19 inactive and IGF2 biallelically active. This can arise either through loss of heterozygosity (LOH) or by a non-LOH pathway involving localized biallelic hypermethylation of H19 DNA. Conversion to this bipaternal endpoint has recently been found to affect not only these two genes, but also at least one other imprinted 11p15.5 gene, KIP2. Since 11p15.5 LOH and biallelic H19 hypermethylation can occur both early and late in tumor progression and since early loss is not associated with bilaterality or multifocality of WTs, these types of lesions appear to be permissive rather than rate-limiting in Wilms' tumorigenesis.

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.

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.

Infrequency of p53 gene mutations in ependymomas.

Ependymomas, which comprise 5% of central nervous system tumors, have not been extensively characterized genetically. The p53 tumor suppressor gene is frequently mutated in human cancer, and is important in the pathogenesis of other central nervous system (CNS) tumors. Chromosomal DNA corresponding to the p53 tumor suppressor gene was amplified by the polymerase chain reaction (PCR) from 31 archival ependymoma specimens. DNA was screened for the presence of p53 mutations by single strand conformational polymorphism (SSCP) analysis; samples with altered mobility were further tested for the presence of mutation by direct DNA sequence analysis. Of the 31 ependymomas tested, one contained a detectable DNA sequence change in the p53 gene. Sequencing revealed a silent mutation in exon 6, at codon 213, which represents a known p53 sequence polymorphism. These finding suggest that in contrast to many other human cancers, p53 mutation is not important in the pathogenesis or progression of ependymomas.

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.

Differential induction of the mitogen-activated protein kinase pathway by bacterial lipopolysaccharide in cultured monocytes and astrocytes.

We recently reported that cyclic AMP (cAMP) specifically inhibits lipopolysaccharide (LPS)-induced interleukin 1 beta (IL-1 beta) transcription initiation in astrocytic cells but enhances the LPS induction of IL-1 beta in monocytic cells. The purpose of this study was to determine how cAMP differentially regulates LPS-induced IL-1 beta transcription in these two cell types. Two essential components of the mitogen-activated protein (MAP) kinase signal-transduction pathway, extracellular-signal-regulated kinase (ERK2; p41 mapk) and Raf-1, have been shown to be targets of LPS stimulation in other cell types, and therefore may be linked to the regulation of IL-1 beta transcription. In the human astrocytic cell line, U-373MG, LPS was found to strongly activate (and cAMP to inhibit) both ERK2 and Raf-1. In the human monocytic cell line, THP-1, LPS minimally activated ERK2 and did not activate Raf-1. These findings suggest that, in astrocytic cells, elevated intracellular cAMP levels may negatively regulate LPS activation of IL-1 beta via the MAP kinase signalling pathway. In contrast, this pathway is not significantly activated by LPS in monocytic cells, thus inhibition by elevated intracellular cAMP levels would not affect IL-1 beta transcription.

Modulation of expression of genes involved in the inflammatory response by lipopolysaccharide and temperature in cultured human astroglial cells.

In bacterial sepsis and meningitis, large concentrations of interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) correlate directly with morbidity and mortality. This laboratory has reported previously that elevated temperature in the physiologic range is associated with down regulation of IL-1 beta and TNF alpha expression in cultured astroglia after lipopolysaccharide (LPS) stimulation. To further investigate the role of elevated temperature in the CNS inflammatory response, the effects of LPS and elevated temperature on the expression of genes that participate in the inflammatory response were determined in cultured transformed human fetal astrocytes and in an astrocytoma cell line. The effect of physiologic temperature elevation on cytokine concentrations in cerebrospinal fluid (CSF) was also investigated in a rabbit meningitis model. The findings indicate that astrocytes express a wide variety of cytokines, growth factors, growth factor receptors, and other genes that could play important roles in CNS inflammation. Furthermore, temperature elevation in the febrile range can lead to alterations in the patterns of expression of many genes involved in the inflammatory response of these cells.

Prolonged in vivo hypoxia enhances nitric oxide synthase type I and type III gene expression in adult rat lung.

Prolonged hypoxia in the adult rat causes a decline in endothelium-derived nitric oxide (NO) production in the pulmonary circulation. To evaluate whether this is related to a decrease in endothelial NO synthase (NOS-III) expression, we determined the effects of hypobaric hypoxia (7 or 21 days) on NOS-III gene expression in adult rat lung. Neuronal NOS (NOS-I) expression was also examined; NOS-I has been immunohistochemically localized to rat bronchiolar epithelium. NOS-III and NOS-I mRNA abundance were assessed in reverse transcription-polymerase chain reaction assays and the proteins were evaluated by immunoblot analysis. After 7 and 21 days of hypoxia, there were increases in the steady-state levels of both NOS-III and NOS-I mRNA, rising 2.7- to 3.0-fold and 2.5- to 2.8-fold, respectively. These findings were confirmed by Northern analyses. In parallel, NOS-III and NOS-I protein abundance were also increased with hypoxia by 3.0- to 3.5-fold and 2.4- to 3.0-fold, respectively. NOS activity detected by [3H]arginine to [3H]citrulline conversion rose 109%. Thus, prolonged in vivo hypoxia causes enhancement of NOS-III and NOS-I gene expression in adult rat lung, indicating that the pulmonary expression of these genes is modulated in vivo. The increase in NOS-III expression does not explain the declines in pulmonary endothelial NO production previously observed following prolonged hypoxia in this model. Alternatively, the fall in NO production may be related to diminished NOS co-factor availability.

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.

Protein synthesis-dependent induction of interleukin-1 beta by lipopolysaccharide is inhibited by dexamethasone via mRNA destabilization in human astroglial cells.

Dexamethasone inhibits lipopolysaccharide-induced synthesis of the cytokine, interleukin-1 beta, in cerebrospinal fluid of patients with bacterial meningitis. Along with monocytes, astrocytes are capable of producing lipopolysaccharide-induced interleukin-1 beta in the central nervous system. The objective of this study was to investigate the induction of interleukin-1 beta mRNA by lipopolysaccharide, and the inhibition of this process by dexamethasone, in human astrocytes using the astrocytoma cell line U-373MG as a model system. Dexamethasone-mediated inhibition of induction of interleukin-1 beta mRNA by lipopolysaccharide required a functional glucocorticoid receptor. In contrast to monocytes, lipopolysaccharide induction of interleukin-1 beta mRNA in U-373MG cells required de novo protein synthesis. Dexamethasone also had no effect on lipopolysaccharide-induced interleukin-1 beta transcriptional initiation in U-373MG cells. U-373MG cells were similar to monocytes, however, with respect to the ability of dexamethasone to decrease interleukin-1 beta mRNA half-life. These findings demonstrate that the mode of lipopolysaccharide induction of interleukin-1 beta mRNA, and inhibition of this process by dexamethasone, can vary in different cell types.

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.

p53 mutations, O6-alkylguanine DNA alkyltransferase activity, and sensitivity to procarbazine in human brain tumors.

BACKGROUND: In human brain tumors, sensitivity to procarbazine as measured by sensitivity in a xenograft tumor model correlated inversely with amounts of the DNA repair enzyme O6-alkylguanine DNA alkyltransferase (AT). METHODS: To test the hypothesis that mutations of the p53 tumor suppressor gene in human tumors also can correlate with the response to chemotherapy, p53 mutations2 were identified in primary human malignant brain tumors and cell lines in which AT activity and procarbazine sensitivity in a xenograft model was ascertained. RESULTS: Mutations were identified in 7 of 21 (33%) specimens tested. Specimens containing p53 mutations tended to exhibit an increased growth delay in procarbazine-treated xenografts and lower amounts of AT. CONCLUSIONS: p53 mutations in brain tumors may contribute to procarbazine sensitivity by failing to induce arrest at the G1/S cell-cycle checkpoint, thereby preventing the repair of procarbazine-induced genetic alterations.