Biomedical subjects

E J Stanbridge

Publications and source records attributed to E J Stanbridge.

At least 19 recordsLinked to original sources

PubMed2006

Identification of a tumor suppressive critical region mapping to 3p14.2 in esophageal squamous cell carcinoma and studies of a candidate tumor suppressor gene, ADAMTS9.

A gene critical to esophageal cancer has been identified. Functional studies using microcell-mediated chromosome transfer of intact and truncated donor chromosomes 3 into an esophageal cancer cell line and nude mouse tumorigenicity assays were used to identify a 1.61 Mb tumor suppressive critical region (CR) mapping to chromosome 3p14.2. This CR is bounded by D3S1600 and D3S1285 microsatellite markers. One candidate tumor suppressor gene, ADAMTS9, maps to this CR. Further studies showed normal expression levels of this gene in tumor-suppressed microcell hybrids, levels that were much higher than observed in the recipient cells. Complete loss or downregulation of ADAMTS9 gene expression was found in 15 out of 16 esophageal carcinoma cell lines. Promoter hypermethylation was detected in the cell lines that do not express this gene. Re-expression of ADAMTS9 was observed after demethylation drug treatment, confirming that hypermethylation is involved in gene downregulation. Downregulation of ADAMTS9 was also found in 43.5 and 47.6% of primary esophageal tumor tissues from Hong Kong and from the high-risk region of Henan, respectively. Thus, this study identifies and provides functional evidence for a CR associated with tumor suppression on 3p14.2 and provides the first evidence that ADAMTS9, mapping to this region, may contribute to esophageal cancer development.

ADAM Proteins↗

PubMed2006

Comparison between pimonidazole binding, oxygen electrode measurements, and expression of endogenous hypoxia markers in cancer of the uterine cervix.

BACKGROUND: Although tumor hypoxia has been associated with a more aggressive phenotype and lower cure rate, there is no consensus as to the method best suited for routine measurement. Binding of the chemical hypoxia marker, pimonidazole, and expression of the endogenous hypoxia markers HIF-1alpha and CAIX were compared for their ability to detect hypoxia in tumor biopsies from 67 patients with advanced carcinoma of the cervix. METHODS: Two biopsies were taken one day after administration of pimonidazole and were analyzed for pimonidazole binding using flow cytometry or immunohistochemistry. CAIX and HIF-1alpha expression and degree of colocalization were measured in sequential antibody-stained sections. Patient subsets were examined for tumor oxygen tension using an Eppendorf electrode, S phase DNA content, or change in HIF-1alpha expression over the course of treatment. RESULTS: Approximately 6% of the tumor area stained positive for pimonidazole, HIF-1alpha, or CAIX. The CAIX positive fraction correlated with the pimonidazole positive fraction (r = 0.60). Weaker but significant correlations were observed between pimonidazole and HIF-1alpha (r = 0.31) and CAIX and HIF-1alpha (r = 0.41). Taking the extent of marker colocalization into consideration increased the confidence that all markers were identifying hypoxic regions. Over 65% of stained areas showed a high degree of colocalization with the other markers. Oxygen microelectrode measurements and S phase fraction were not correlated with the hypoxic fraction measured using the three hypoxia markers. HIF-1alpha levels tended to decrease with time after the start of therapy. CONCLUSIONS: Endogenous hypoxia marker binding shows reasonable agreement, in extent and location, with binding of pimonidazole. CAIX staining pattern is a better match to the pimonidazole staining pattern than is HIF-1alpha, and high CAIX expression in the absence (or low levels) of HIF-1alpha may indicate a different biology.

Antigens, Neoplasm↗

PubMed2005

STRA13 expression and subcellular localisation in normal and tumour tissues: implications for use as a diagnostic and differentiation marker.

BACKGROUND: STRA13 is a bHLH transcription factor that plays a crucial role in cell differentiation, proliferation, apoptosis, and response to hypoxia. OBJECTIVE: To assess STRA13 involvement in carcinogenesis and evaluate its diagnostic value. METHODS: A comprehensive analysis was undertaken of the endogenous protein expression in 389 normal and corresponding malignant specimens, using newly generated polyclonal antibodies. RESULTS: STRA13 was commonly expressed in epithelial cells of normal and neoplastic tissues where it was confined mostly to the nucleus. Intense cytoplasmic STRA13 immunoreactivity was characteristic of myoepithelial and differentiated squamous epithelial cells of all organ sites and their neoplastic counterparts, suggesting application of STRA13 as a myoepithelial cell marker. A distinctive apical granular cytoplasmic staining pattern observed in the pancreas and large intestine was retained in corresponding metastatic carcinomas, providing for identification of the primary sites of these disseminating tumours. In less differentiated tumours there was a tendency to lose the cytoplasmic staining or to switch to nuclear STRA13 staining. Analysis of STRA13, HIF-1alpha, and CAIX expression patterns in a large set of various tumours substantiated the association of STRA13 with HIF-1alpha expression and hypoxia in vivo. Investigation of the molecular mechanisms of STRA13 nucleo-cytoplasmic shuttling suggested that STRA13 employs nuclear import/export that utilises the NLS/NES motifs situated within the N-terminus and in the middle of the protein. CONCLUSIONS: STRA13 may serve as a marker for myoepithelial cells, for the degree of tumour differentiation, and for identification of the primary site of certain metastatic tumours. In combination with CAIX and CAXII markers, it may lead to a more accurate classification of all renal carcinomas.

Antibody Specificity↗

PubMed2005

Intratumoral oxygenation of invasive squamous cell carcimoma of the vulva is not correlated with regional lymph node metastasis.

INTRODUCTION: Tumour hypoxia has been found to be associated with tumour aggressiveness. Our primary aim was to explore the relationship between pretreatment tumour oxygenation in primary vulvar carcinoma and nodal status. Our secondary objective was to assess if there was a relationship between the clinical and biological variables. METHODS: 20 women with ISCC of the vulva were assessed with pretreatment primary tumour oxygenation with an Eppendorf pO2 probe. Patients underwent standard surgical management. Pathological assessment of the primary and nodal tissues was then performed. Primary tumour specimens were also stained for microvessel density and carbonic anhydrase IX. The relationship between smoking, preoperative Hgb, tumour CAIX expression, MVD, and Eppendorf pO2 measurements vs nodal metastasis and between these clinical and biological variables was assessed. RESULTS: Seven patients had positive lymph nodes, 13 had negative nodes. While neither current smoking status, tumour size, tumour oxygen measurements, MVD and CAIX expression correlated with metastatic nodal disease, a low preoperative Hgb correlated with pathological nodal status (p < 0.027). CONCLUSIONS: Although this analysis failed to demonstrate a strong correlation between various measures of tumour oxygenation with nodal metastasis, it may be due to the small number of patients. Only preoperative anaemia is correlated with nodal metastasis in early ISCC of the vulva.

Aged↗

PubMed2003

Expression of cell surface transmembrane carbonic anhydrase genes CA9 and CA12 in the human eye: overexpression of CA12 (CAXII) in glaucoma.

PURPOSE: Carbonic anhydrase enzymes (CAs) are universally involved in many fundamental physiological processes, including acid base regulation and fluid formation and movement. In glaucoma patients, CA inhibitors are very effective in lowering intraocular pressure by reducing the rate of aqueous humour secretion mediated by the CAs in the ciliary epithelium. In this work, we investigated the expression and tissue distribution of two recently discovered CA genes CA9 (CAIX) and CA12 (CAXII) in fetal, neonatal, and adult human eyes with and without glaucoma. METHODS: CAIX and CAXII expression in 16 normal and 10 glaucomatous eyes, and in cultured non-pigmented ciliary epithelial cells (NPE) from normal and glaucoma eye donors was assessed by immunostaining. In addition, northern blot hybridisation was performed to assess expression of CA4, CA9, and CA12 mRNA in cultured NPE cells from normal and glaucoma donors. RESULTS: CAXII was localised primarily to the NPE with its expression prominent during embryonic eye development but which decreased significantly in adults. CAIX expression in the NPE was very low. The epithelium of cornea and lens occasionally expressed both enzymes at low levels during development and in adult eye, and no expression was detected in the retina. The NPE from glaucoma eyes expressed higher levels of CAXII, but not CAIX, in comparison with normal eyes. This expression pattern was retained in cultured NPE cell lines. NPE cells from a glaucoma patient showed a five-fold increase in the CA12 mRNA level with no detectable expression of CA9 mRNA. Also, no expression of the CA4 gene encoding a GPI anchored plasma membrane protein was detected on these northern blots. CONCLUSIONS: Transmembrane CAIX and CAXII enzymes are expressed in the ciliary cells and, thus, may be involved in aqueous humour production. CA12 may be a targeted gene in glaucoma.

Blotting, Northern↗

PubMed2001

Carbonic anhydrase 9 as an endogenous marker for hypoxic cells in cervical cancer.

The presence of radiation-resistant hypoxic cells in some solid tumors is known to predict for relapse after radiotherapy. Use of an endogenous marker of hypoxia would be a convenient alternative to current methods that measure tumor oxygenation, provided the marker could be shown to reliably identify viable, radiation-resistant, hypoxic cells. Carbonic anhydrase 9 (CA9) is a transmembrane protein overexpressed in a wide variety of tumor types and induced by hypoxia. Using a monoclonal antibody and cell sorting, CA9-positive cells in SiHa cervical carcinoma xenografts growing in immunodeficient mice were found to be clonogenic, resistant to killing by ionizing radiation, and preferentially able to bind the hypoxia marker pimonidazole. CA9 and pimonidazole immunostaining were compared in formalin-fixed sections from tumors of 18 patients undergoing treatment for cancer of the cervix. Excellent colocalization was observed, although the area of the tumor section that bound anti-CA9 antibodies represented double the number of cells that bound anti-pimonidazole antibodies. Occasional regions staining with pimonidazole but not CA9 could be indicative of transient changes in tumor perfusion. Results support the hypothesis that CA9 is a useful endogenous marker of tumor hypoxia.

Animals↗

PubMed2001

Characterization of the MN/CA 9 promoter proximal region: a role for specificity protein (SP) and activator protein 1 (AP1) factors.

MN/CA IX (MN) is a tumour-associated isoenzyme of the carbonic anhydrase family. Previous deletion analysis of the MN promoter established that protected regions (PRs) 1 and 2 are crucial for its transcriptional activity. Computer-assisted searching indicated putative binding sites for activator protein (AP) 2 and specificity protein (SP) 1 transcription factors, plus a CACCC box in PR1 and an AP1 site in PR2. PR1 produced four complexes in electrophoretic mobility-shift assay (EMSA) with HeLa nuclear extracts. Of these, three were completely competed with the SP1 and transforming growth factor-beta retinoblastoma control-element CACCC box (RCE) probes, whereas the AP2 probe competed against the same three complexes partially. Supershift EMSA identified SP1 in the complex 1 and SP3 in the complexes 2 and 4. Point mutations in the SP1 site abrogated the PR1 function, while mutations affecting the overlapping CACCC box/AP2 site in PR1 had minor effect on MN promoter activity. Block-replaced MN promoter mutants that had a consensus binding site (SP1 or AP2) or the RCE in place of PR1 demonstrated the stringent selectivity of the PR1 position as only the SP1 mutant reconstituted the MN promoter activity. The consensus SP1 probe generated the same SP1 and SP3 complexes as PR1 in EMSA; therefore we conclude that SP activity is both necessary and sufficient in the PR1 position. The critical role of AP1 in the PR2 position was confirmed by supershift of the PR2 complex with c-Fos antibody and markedly decreased activity of the construct with a mutated AP1 site. Detailed deletion analysis proved that PR1+PR2 account for 90% of the MN promoter activity, while neither PR1 nor PR2 on their own are sufficient for transactivation. Thus, synergistic co-operation between SP and AP1 factors bound to the adjacent PR1 and PR2, respectively, is necessary for MN transcriptional activity. The PR1+PR2 module also stimulated transcription from a heterologous promoter. The modulation of AP1 activity with PMA stimulated MN expression and activated the MN promoter, whereas inhibition of protein kinase C activity had no effect on MN expression in HeLa cells.

Antigens, Neoplasm↗

PubMed2001

Evidence for a putative senescence gene locus within the chromosomal region 10p14-p15.

High-risk human papillomavirus (HPV) types 16 and 18 are involved in the multistep process of cervical cancer. Transfection of normal keratinocytes with high-risk HPV-DNA generally gives rise to immortal cultures. This may be explained by the loss of senescence genes as a consequence of HPV-induced genetic instability. On the basis of the dominance of cellular senescence over immortality, fusion of normal keratinocytes with HPV-immortalized cells results in complementation of these putative gene defects. In a previous study, we showed that underrepresentation of chromosome 10 is a characteristic phenomenon during the early phase of immortalization. Here we show that introduction of a normal copy of chromosome 10 into HPV16-immortalized cells (HPKII) by Microcell-mediated chromosome transfer resulted in senescence of a significant number of hybrids. By using several derivatives of chromosome 10 for further fusion experiments, the chromosomal region responsible for senescence could be assigned to 10p14-p15. The potential significance of loss of gene function in this region is underlined by the high frequency (38.7%) of loss of heterozygosity in cervical cancers including early stage tumors.

Carcinoma, Squamous Cell↗

PubMed2001

RBP1L1, a retinoblastoma-binding protein-related gene encoding an antigenic epitope abundantly expressed in human carcinomas and normal testis.

BACKGROUND: Antibodies isolated from cancer patients have been used to identify genes encoding tumor-associated antigen epitopes relevant to immune responses in cancer patients. In this report, we used an immunoglobulin G (IgG) purified from serum of a patient with breast cancer to identify its corresponding epitope, gene, and protein-retinoblastoma-binding protein-1-like protein-1 (RBP1L1)-and determined whether it is a potential molecular marker for various cancers. METHODS: IgG purified from the serum of a patient with breast cancer was used to screen an MCF-7 breast cancer cell complementary DNA (cDNA) expression library for immunoreactive clones. The cDNAs identified were cloned and sequenced. Immunoreactivity of specific amino acids in the epitope was determined by western blot analysis and enzyme-linked immunosorbent assay. The cellular location of the antigen was determined by immunoperoxidase staining with purified RBP1L1-specific IgG. Gene expression in various human carcinomas and normal tissues was examined by northern blot analysis and the reverse transcription-polymerase chain reaction. RESULTS: Our purified IgG recognized just one epitope on RBP1L1. The complete 5802-base-pair RBP1L1 cDNA encodes a 1226-amino acid protein containing the antigenic epitope IKPSLGSKK. The derived protein sequence of RBP1L1 shares 74% and 37% amino acid identity, respectively, with a partial sequence of the retinoblastoma-binding protein and the complete sequence of retinoblastoma-binding protein-1. The RBP1L1 epitope was localized to the cytoplasm of MCF-7 cells but was not detected in peripheral blood mononuclear cells. High expression of RBP1L1 messenger RNA was found in human breast, lung, colon, pancreatic, and ovarian cancers and in normal testis, but expression was limited in other normal tissues. CONCLUSIONS: RBP1L1 appears to be a molecular marker associated with a broad range of human malignancies.

Biomarkers, Tumor↗

PubMed2001

Link of a new type of apoptosis-inducing gene ASY/Nogo-B to human cancer.

Although apoptosis plays an essential role in the embryogenesis and homeostasis of multicellular organisms, this mechanism has not yet been fully clarified. We isolated a novel human apoptosis-inducing gene, ASY, which encodes an endoplasmic reticulum-targeting protein without any known apoptosis-related motifs. This gene is identical to the Nogo-B, a splice variant of the Nogo-A which has recently been shown to be an inhibitor of neuronal regeneration in the central nervous system. Ectopic expression of the ASY gene led to extensive apoptosis, particularly in cancer cells. Furthermore, transcription of the ASY gene was suppressed in small cell lung cancer. These results suggest that a new type of apoptosis-inducing gene, namely, ASY, may be involved in the development of certain types of cancer.

Amino Acid Motifs↗

PubMed2001

Mechanisms of differential activation of target gene promoters by p53 hinge domain mutants with impaired apoptotic function.

Suppression of tumor cell growth by p53 results from the activation of both apoptosis and cell cycle arrest functions that have been shown to be separable activities of p53. We report here that some mutants in the p53 hinge domain, a short linker between the DNA binding and tetramerization domains, differentially activated the promoters of p53 target genes and possessed an impaired apoptotic function. Our results indicate that the hinge domain may play an important role in differentially regulating p53 cell cycle arrest and apoptotic functions. However, the mechanisms by which p53 hinge domain mutants differentially activate its target genes, e.g. p21(WAF1/CIP1) and Bax, remain unknown. To investigate the possible mechanisms, recombinant p21(WAF1/CIP1) and Bax promoters were constructed, resulting in rearrangement of the existing p53 binding sites within a given promoter or actually swapping p53 binding sites between the two promoters. Our results suggest that multiple mechanisms of differential transactivation occur, depending on the molecular nature of the relevant hinge domain mutant, such as the possibility that dual separate DNA binding sites in the p21(WAF1/CIP1) promoter are responsible for the selective transactivation activity of p53 hinge domain mutant del300-327, which has a large deletion in the hinge domain. Lack of ideal p53 binding sites in the Bax promoter results in less potent activation than that seen with the p21(WAF1/CIP1) promoter when it is transactivated by hinge domain point mutant mutR306P or short deletion mutant del300-308 proteins. How the single mutation or the short deletion affect the conformation of p53 and consequently the transactivation of the Bax promoter will require further investigation of the relevant p53 protein: DNA-binding domain by NMR and x-ray crystallographic techniques.

Apoptosis↗

PubMed2001

The candidate tumor suppressor gene, RASSF1A, from human chromosome 3p21.3 is involved in kidney tumorigenesis.

Clear cell-type renal cell carcinomas (clear RCC) are characterized almost universally by loss of heterozygosity on chromosome 3p, which usually involves any combination of three regions: 3p25-p26 (harboring the VHL gene), 3p12-p14.2 (containing the FHIT gene), and 3p21-p22, implying inactivation of the resident tumor-suppressor genes (TSGs). For the 3p21-p22 region, the affected TSGs remain, at present, unknown. Recently, the RAS association family 1 gene (isoform RASSF1A), located at 3p21.3, has been identified as a candidate lung and breast TSG. In this report, we demonstrate aberrant silencing by hypermethylation of RASSF1A in both VHL-caused clear RCC tumors and clear RCC without VHL inactivation. We found hypermethylation of RASSF1A's GC-rich putative promoter region in most of analyzed samples, including 39 of 43 primary tumors (91%). The promoter was methylated partially or completely in all 18 RCC cell lines analyzed. Methylation of the GC-rich putative RASSF1A promoter region and loss of transcription of the corresponding mRNA were related causally. RASSF1A expression was reactivated after treatment with 5-aza-2'-deoxycytidine. Forced expression of RASSF1A transcripts in KRC/Y, a renal carcinoma cell line containing a normal and expressed VHL gene, suppressed growth on plastic dishes and anchorage-independent colony formation in soft agar. Mutant RASSF1A had reduced growth suppression activity significantly. These data suggest that RASSF1A is the candidate renal TSG gene for the 3p21.3 region.

Azacitidine↗

PubMed2001

Detection of differentially expressed genes in HeLa x fibroblast hybrids using subtractive suppression hybridization.

To understand genetic differences and similarities between tumorigenic and nontumorigenic HeLa x fibroblast hybrid cells, subtractive suppression hybridization (SSH), based on suppression PCR and a combination of normalization and subtraction in a single procedure, was used. Using the nontumorigenic CGL1 and tumorigenic CGL3, forward (CGL1-CGL3) and reverse (CGL3-CGL1) subtracted libraries were constructed. Among 192 clones, seven were identified as differentially expressed genes specific for either CGL1 or CGL3. All seven were not reported previously as differentially expressed genes in this hybrid system. In the forward subtraction, p16 was isolated, indicating the involvement of the loss of tumorigenic phenotype. Subsequent transfection of wild-type p16 to the tumorigenic CGL3 showed growth suppression in colony formation assay; however, no tumor suppression was observed when the transfectant was inoculated into nude mice. These results indicate that: (a) SSH is a suitable method to identify differentially expressed genes in two types of cells; and (b) although p16 plays some roles in growth suppression, the p16-transfected CGL3 is still capable to proliferate in vivo.

Animals↗

PubMed2001

Oligonucleotide microarray expression analysis of genes whose expression is correlated with tumorigenic and non-tumorigenic phenotype of HeLa x human fibroblast hybrid cells.

In order to understand the differences and similarities between tumorigenic and non-tumorigenic HeLaxhuman fibroblast hybrids, gene expression profiles were examined with synthetic oligonucleotide arrays containing nearly 7000 gene probe sets. We used two pairs of genetically related hybrids, each pair representing individual clones of non-tumorigenic and tumorigenic segregant hybrids, respectively. Analysis of six possible comparisons, utilizing two algorithms, identified 204 genes with differential expression. The greater number of differentially expressed genes was observed when non-tumorigenic hybrids were compared with tumorigenic segregants. Fifteen and 14 genes, respectively, were consistently found to be differentially expressed in non-tumorigenic and tumorigenic cells. Among those 29 differentially expressed genes, three (intestinal alkaline phosphatase, caveolin-1, and solute carrier family2, member3) have been reported previously to be associated with the tumorigenic phenotype, using the same hybrid pairs. In addition, among the genes previously detected by differential display, 78% of them exhibited more than 5-fold change, demonstrating a high consistency between the two methods of differential gene expression. These findings suggest that synthetic oligonucleotide arrays are a powerful and highly reproducible tool to identify those genes whose expression is associated with certain phenotypes.

Algorithms↗

PubMed2001

Expression of hypoxia-inducible cell-surface transmembrane carbonic anhydrases in human cancer.

An acidic extracellular pH is a fundamental property of the malignant phenotype. In von Hippel-Lindau (VHL)-defective tumors the cell surface transmembrane carbonic anhydrase (CA) CA9 and CA12 genes are overexpressed because of the absence of pVHL. We hypothesized that these enzymes might be involved in maintaining the extracellular acidic pH in tumors, thereby providing a conducive environment for tumor growth and spread. Using Northern blot analysis and immunostaining with specific antibodies we analyzed the expression of CA9 and CA12 genes and their products in a large sample of cancer cell lines, fresh and archival tumor specimens, and normal human tissues. Expression was also analyzed in cultured cells under hypoxic conditions. Expression of CA IX and CA XII in normal adult tissues was detected only in highly specialized cells and for most tissues their expression did not overlap. Analysis of RNA samples isolated from 87 cancer cell lines and 18 tumors revealed high-to-moderate levels of expression of CA9 and CA12 in multiple cancers. Immunohistochemistry revealed high-to-moderate expression of these enzymes in various normal tissues and multiple common epithelial tumor types. The immunostaining was seen predominantly on the cell surface membrane. The expression of both genes was markedly induced under hypoxic conditions in tumors and cultured tumor cells. We conclude that the cell surface trans-membrane carbonic anhydrases CA IX and CA XII are overexpressed in many tumors suggesting that this is a common feature of cancer cells that may be required for tumor progression. These enzymes may contribute to the tumor microenvironment by maintaining extracellular acidic pH and helping cancer cells grow and metastasize. Our studies show an important causal link between hypoxia, extracellular acidification, and induction or enhanced expression of these enzymes in human tumors.

Biomarkers, Tumor↗

PubMed2001

Paired human fibrosarcoma cell lines that possess or lack endogenous mutant N-ras alleles as experimental model for Ras signaling pathways.

We present here a human cell model for examination of mutant N-ras function. The HT1080 human fibrosarcoma cell line is pseudodiploid and contains a single endogenous mutant N-ras allele. MCH603c8 cells are a variant of HT1080 cells, in which the mutant allele has been deleted. The two cell lines differ dramatically in the constitutive levels of activation of downstream members of the Ras signaling pathways, and in biological features of transformation and tumorigenicity. Downregulation or activation of individual Ras-dependent pathways can be accomplished via transfection of dominant negatives or activated mutant cDNAs into HT1080 and MCH603c8 cells, respectively. The biochemical and biological consequences of expression of these mutant cDNAs can be assessed. There are dramatic effects on both the transformed and tumorigenic phenotype, depending on the cell line and mutant cDNA that is transfected.

Actins↗

PubMed2001

Role of phosphoinositide 3-kinase in the aggressive tumor growth of HT1080 human fibrosarcoma cells.

We have developed a model system of human fibrosarcoma cell lines that do or do not possess and express an oncogenic mutant allele of N-ras. HT1080 cells contain an endogenous mutant allele of N-ras, whereas the derivative MCH603 cell line contains only wild-type N-ras. In an earlier study (S. Gupta et al., Mol. Cell. Biol. 20:9294-9306, 2000), we had shown that HT1080 cells produce rapidly growing, aggressive tumors in athymic nude mice, whereas MCH603 cells produced more slowly growing tumors and was termed weakly tumorigenic. An extensive analysis of the Ras signaling pathways (Raf, Rac1, and RhoA) provided evidence for a potential novel pathway that was critical for the aggressive tumorigenic phenotype and could be activated by elevated levels of constitutively active MEK. In this study we examined the role of phosphoinositide 3-kinase (PI 3-kinase) in the regulation of the transformed and aggressive tumorigenic phenotypes expressed in HT1080 cells. Both HT1080 (mutant N-ras) and MCH603 (wild-type N-ras) have similar levels of constitutively active Akt, a downstream target of activated PI 3-kinase. We find that both cell lines constitutively express platelet-derived growth factor (PDGF) and PDGF receptors. Transfection with tumor suppressor PTEN cDNA into HT1080 and constitutively active PI 3-kinase-CAAX cDNA into MCH603 cells, respectively, resulted in several interesting and novel observations. Activation of the PI 3-kinase/Akt pathway, including NF-kappaB, is not required for the aggressive tumorigenic phenotype in HT1080 cells. Activation of NF-kappaB is complex: in MCH603 cells it is mediated by Akt, whereas in HT1080 cells activation also involves other pathway(s) that are activated by mutant Ras. A threshold level of activation of PI 3-kinase is required in MCH603 cells before stimulatory cross talk to the RhoA, Rac1, and Raf pathways occurs, without a corresponding activation of Ras. The increased levels of activation seen were similar to those observed in HT1080 cells, except for Raf and MEK, which were more active than HT1080 levels. This cross talk results in conversion to the aggressive tumorigenic phenotype. This latter observation is consistent with our previous observation that overstimulation of the activity of endogenous members of Ras signaling pathways, activated MEK in particular, is a prerequisite for aggressive tumorigenic growth.

Actins↗

PubMed2001

Examination of the oncogenic potential of H19 gene in HeLa x normal human fibroblast hybrid cells.

Fusion of tumorigenic HeLa cells with human skin fibroblasts results in chromosomally stable non-tumorigenic hybrids. The studies of rare spontaneous tumorigenic segregants from the non-tumorigenic hybrid have implicated the loss of one copy of human fibroblast chromosome 11 with concomitant re-expression of tumorigenicity. In a previous study of differential display screening, we reported the tumorigenic-specific expression of H19 gene as a possible candidate for elicitation of tumorigenic phenotype. In this study, we examined the expression of H19 gene using gamma-ray-induced tumorigenic mutants (GIMs) and non-tumorigenic irradiated control cells (CONs) from the non-tumorigenic hybrids. H19 expression was recognized in all the GIMs with various expression levels, whereas no CONs expressed the H19 gene. To examine the tumorigenic potentials of H19 gene directly, we introduced an H19 gene expression vector into the non-tumorigenic hybrids and assayed the tumorigenicity of the transfectants by s.c. injection into athymic nude mice. However, no transfectants with stable H19 gene expression induced in vivo tumor growth. These results suggest that expression of the H19 gene may be necessary but is not sufficient to confer the tumorigenic phenotype in HeLa x fibroblast hybrids.

Animals↗