Search PubMed⌕ Search

Biomedical subjects

Latha Ramdas

Publications and source records attributed to Latha Ramdas.

16 recordsLinked to original sources
PubMed2006

Mitochondrial respiration defects in cancer cells cause activation of Akt survival pathway through a redox-mediated mechanism.

Cancer cells exhibit increased glycolysis for ATP production due, in part, to respiration injury (the Warburg effect). Because ATP generation through glycolysis is less efficient than through mitochondrial respiration, how cancer cells with this metabolic disadvantage can survive the competition with other cells and eventually develop drug resistance is a long-standing paradox. We report that mitochondrial respiration defects lead to activation of the Akt survival pathway through a novel mechanism mediated by NADH. Respiration-deficient cells (rho(-)) harboring mitochondrial DNA deletion exhibit dependency on glycolysis, increased NADH, and activation of Akt, leading to drug resistance and survival advantage in hypoxia. Similarly, chemical inhibition of mitochondrial respiration and hypoxia also activates Akt. The increase in NADH caused by respiratory deficiency inactivates PTEN through a redox modification mechanism, leading to Akt activation. These findings provide a novel mechanistic insight into the Warburg effect and explain how metabolic alteration in cancer cells may gain a survival advantage and withstand therapeutic agents.

Apoptosis↗
PubMed2006

Early effects of imatinib mesylate on the expression of insulin-like growth factor binding protein-3 and positron emission tomography in patients with gastrointestinal stromal tumor.

BACKGROUND: Imatinib has demonstrated marked clinical efficacy against gastrointestinal stromal tumor (GIST). Microarray technology, real-time polymerase chain reaction (PCR) validation, and fluorodeoxyglucose-positron emission tomography (FDG-PET) imaging were used to study the early molecular effects of imatinib antitumor activity in GIST. METHODS: After exposure of sensitive and resistant sarcoma cell lines to imatinib for 24 to 48 hours, the changes in gene expression were evaluated using a 1146 unique pathway array with Western blot validation. Real-time PCR was used to confirm changes in gene expression in human GIST samples (preimatinib biopsy and postimatinib surgical specimen after 3-7 days of therapy). FDG-PET was performed to correlate radiographic findings with the effects of imatinib on gene expression in GIST. RESULTS: In all, 55 genes demonstrated a > or = 2-fold change after imatinib treatment of the GIST882 cells. Among these genes there was up-regulation of insulin-like growth factor binding protein-3 (IGFBP-3), a protein that modulates proliferation and apoptosis. Western blot analysis confirmed the increase of IGFBP-3 only in imatinib-sensitive GIST882 cells. Up to a 7-fold induction (49% mean increase; P = .08) of IGFBP-3 mRNA was found in tumor samples from patients with low residual FDG uptake, whereas there was an up to 12-fold reduction (-102% mean decrease; P = .03) in IGFBP-3 in those patients with high residual FDG uptake after imatinib therapy. CONCLUSIONS: In the current study, imatinib appears to regulate numerous genes and specifically induces IGFBP-3 in GIST cells and tumor samples. IGFBP-3 levels also were found to be inversely correlated with residual FDG uptake in GIST patients early in imatinib therapy. These initial observations suggest that IGFBP-3 is an important early marker of antitumor activity of imatinib in GIST.

Antineoplastic Agents↗
PubMed2006

Microarray image scanning.

Of the technologies available for measuring gene expression, microarrays using cDNA targets is one of the most common and well-developed high-throughput techniques. With this technique, the expression levels of thousands of genes are measured simultaneously. DNA probes are immobilized on solid surfaces, either membrane-based or chemically coated glass surfaces. On glass arrays, the probes are hybridized with fluorescent-labeled target samples. Fluorescence intensities, which reflect gene expression levels, are detected by imaging the array using a laser or white-light source and capturing the image using photomultiplier tube detection or a charge-coupled device camera. Different laser-based scanners are used in laboratories to scan microarray images. This chapter discusses the imaging process and the protocols being developed.

Carbocyanines↗
PubMed2006

Chemical genetic identification of the IGF-linked pathway that is mediated by STAT6 and MFP2.

Insulin-like growth factor 2 (IGF2) is a potent mitogen whose deregulation plays a role in developing liver, breast, and prostate cancers. Here, we take a small-molecule approach to investigate molecular pathways that modulate IGF2 signaling, by using chromeceptin, a synthetic molecule that selectively impairs the viability and growth of IGF2-overexpressing hepatocellular carcinoma cells. Affinity purification revealed that chromeceptin binds to multifunctional protein 2 (MFP-2), a seemingly multifunctional enzyme implicated in peroxisomal beta-oxidation. The small molecule-protein interaction stimulates the expression of IGF binding protein 1 (IGFBP-1) and suppressor of cytokine signaling-3 (SOCS-3), two cellular attenuators of the IGF signals, through activation of signal transducers and activators of transcription 6 (STAT6). The results underline the importance of STATs in IGF/insulin regulation, and they implicate a new pathway for STAT6 activation that is amenable to small-molecule intervention.

17-Hydroxysteroid Dehydrogenases↗
PubMed2005

Phosphorylation of galectin-3 contributes to malignant transformation of human epithelial cells via modulation of unique sets of genes.

Galectin-3 is a multifunctional beta-galactoside-binding protein implicated in apoptosis, malignant transformation, and tumor progression. The mechanisms by which galectin-3 contributes to malignant progression are not fully understood. In this study, we found that the introduction of wild-type galectin-3 into nontumorigenic, galectin-3-null BT549 human breast epithelial cells conferred tumorigenicity and metastatic potential in nude mice, and that galectin-3 expressed by the cells was phosphorylated. In contrast, BT549 cells expressing galectin-3 incapable of being phosphorylated (Ser6-->Glu Ser6-->Ala) were nontumorigenic. A microarray analysis of 10,000 human genes, comparing BT549 transfectants expressing wild-type and those expressing phosphomutant galectin-3, identified 188 genes that were differentially expressed (>2.5-fold). Genes affected by introduction of wild-type phosphorylated but not phosphomutant galectin-3 included those involved in oxidative stress, a novel noncaspase lysosomal apoptotic pathway, cell cycle regulation, transcriptional activation, cytoskeleton remodeling, cell adhesion, and tumor invasion. The reliability of the microarray data was validated by real-time reverse transcription-PCR (RT-PCR) and by Western blot analysis, and clinical relevance was evaluated by real-time RT-PCR screening of a panel of matched pairs of breast tumors. Differentially regulated genes in breast cancers that are also predicted to be associated with phospho-galectin-3 in transformed BT549 cells include C-type lectin 2, insulin-like growth factor-binding protein 5, cathepsins L2, and cyclin D1. These data show the functional diversity of galectin-3 and suggest that phosphorylation of the protein is necessary for regulation (directly or indirectly) of unique sets of genes that play a role in malignant transformation.

Animals↗
PubMed2005

Molecular signatures associated with clinical outcome in patients with high-risk head-and-neck squamous cell carcinoma treated by surgery and radiation.

PURPOSE: The local-regional control rate for advanced head-and-neck squamous cell carcinoma (HNSCC) remains poor and is unpredictable for a given individual. This study examined whether gene expression patterns developed from tumors from surgicopathologic, criteria-defined, high-risk HNSCC patients could be correlated with clinical outcomes, namely, metastasis or nonrecurrent disease. METHODS AND MATERIALS: Fifteen primary tumors from patients treated with a consistent protocol of surgery followed by radiotherapy were examined. Seven of these tumors were from high-risk patients who developed distant metastasis (DM), and eight tumors were from patients with no recurrence (NR) (median follow-up, 59 months). RESULTS: Unsupervised clustering of gene expression did not separate the two groups from one another, but when supervised methodologies were applied, 205 genes discriminated the two groups. Within the DM group, genes associated with cell growth and proliferation; DNA replication, recombination, and repair; antiapoptotic pathways; cell adhesion; and angiogenesis were identified. For NR samples, discriminatory genes were associated with the onset of apoptosis. CONCLUSIONS: Our data suggest that gene expression analysis of surgically excised HNSCC tumors from patients considered at high risk for recurrence has the potential to identify individuals susceptible to metastasis on the basis of distinct gene-expression patterns. These patients would be ideal candidates for testing systemic therapy.

Carcinoma, Squamous Cell↗
PubMed2004

Combination of 5-fluorouracil and N1,N11-diethylnorspermine markedly activates spermidine/spermine N1-acetyltransferase expression, depletes polyamines, and synergistically induces apoptosis in colon carcinoma cells.

The thymidylate synthase inhibitor 5-fluorouracil (5-FU) is used widely for chemotherapy of colorectal carcinoma. Recent studies showed that 5-FU affects polyamine metabolism in colon carcinoma cells. We therefore examined whether combinations of 5-FU with drugs that specifically target polyamine metabolism, i.e. N1,N11-diethylnorspermine (DENSPM) or alpha-difluoromethylornithine (DFMO), have synergistic effects in killing HCT116 colon carcinoma cells with wild-type or absent p53. Our results showed that simultaneous 5-FU and DENSPM, a spermine analogue, synergistically increased transcript levels of the polyamine catabolism enzyme spermidine/spermine N1-acetyltransferase, depleted spermine and spermidine, increased acetylated spermidine, and produced synergistic tumor cell apoptosis in both p53 wild-type and p53-null variants. By contrast, simultaneous combination of 5-FU with DFMO, an inhibitor of the polyamine biosynthetic enzyme ornithine decarboxylase, depleted putrescine but did not produce synergistic cell killing. Some pre-treatment and post-treatment regimens of DENSPM and DFMO were antagonistic to 5-FU depending on cellular p53 status. Protein and transcriptome expression analysis showed that combined 5-FU and DENSPM treatment activated caspase 9, but not caspase 3, and significantly suppressed NADH dehydrogenases and cytochrome c oxidases, consistent with the observed increase in hydrogen peroxide, loss of mitochondrial membrane potential, and release of cytochrome c. Our findings demonstrate the importance of the polyamine pathway in 5-FU effects and suggest that the combination of 5-FU with DENSPM has potential for development as therapy for colorectal carcinoma.

Acetyltransferases↗
PubMed2004

A missense mutation in KIT kinase domain 1 correlates with imatinib resistance in gastrointestinal stromal tumors.

KIT gain of function mutations play an important role in the pathogenesis of gastrointestinal stromal tumors (GISTs). Imatinib is a selective tyrosine kinase inhibitor of ABL, platelet-derived growth factor receptor (PDGFR), and KIT and represents a new paradigm of targeted therapy against GISTs. Here we report for the first time that, after imatinib treatment, an additional specific and novel KIT mutation occurs in GISTs as they develop resistance to the drug. We studied 12 GIST patients with initial near-complete response to imatinib. Seven harbored mutations in KIT exon 11, and 5 harbored mutations in exon 9. Within 31 months, six imatinib-resistant rapidly progressive peritoneal implants (metastatic foci) developed in five patients. Quiescent residual GISTs persisted in seven patients. All six rapidly progressive imatinib-resistant implants from five patients show an identical novel KIT missense mutation, 1982T-->C, that resulted in Val654Ala in KIT tyrosine kinase domain 1. This novel mutation has never been reported before, is not present in pre-imatinib or post-imatinib residual quiescent GISTs, and is strongly correlated with imatinib resistance. Allelic-specific sequencing data show that this new mutation occurs in the allele that harbors original activation mutation of KIT.

Alleles↗
PubMed2004

Improving signal intensities for genes with low-expression on oligonucleotide microarrays.

BACKGROUND: DNA microarrays using long oligonucleotide probes are widely used to evaluate gene expression in biological samples. These oligonucleotides are pre-synthesized and sequence-optimized to represent specific genes with minimal cross-hybridization to homologous genes. Probe length and concentration are critical factors for signal sensitivity, particularly when genes with various expression levels are being tested. We evaluated the effects of oligonucleotide probe length and concentration on signal intensity measurements of the expression levels of genes in a target sample. RESULTS: Selected genes of various expression levels in a single cell line were hybridized to oligonucleotide arrays of four lengths and four concentrations of probes to determine how these critical parameters affected the intensity of the signal representing their expression. We found that oligonucleotides of longer length significantly increased the signals of genes with low-expression in the target. High-expressing gene signals were also boosted but to a lesser degree. Increasing the probe concentration, however, did not linearly increase the signal intensity for either low- or high-expressing genes. CONCLUSIONS: We conclude that the longer the oligonuclotide probe the better the signal intensities of low expressing genes on oligonucleotide arrays.

Cell Line, Tumor↗
PubMed2004

Signal transducer and activator of transcription-3 activation contributes to high tissue inhibitor of metalloproteinase-1 expression in anaplastic lymphoma kinase-positive anaplastic large cell lymphoma.

The tissue inhibitor of metalloproteinase-1 (TIMP1) is expressed in a subset of malignant lymphomas and can inhibit tumor spread and promote cell survival. Recent data suggest that TIMP1 expression may be regulated by signal transducer and activator of transcription (STAT)-3. Thus, we tested the hypothesis that TIMP1 expression is related to STAT3 activation in lymphomas, with a focus on anaplastic large cell lymphomas (ALCLs), which are known to express high levels of phosphorylated/active STAT3 (pSTAT3). Specific inhibition of STAT3 with a dominant-negative construct led to concentration-dependent down-regulation of TIMP1 expression in two anaplastic lymphoma kinase (ALK)(+) ALCL cell lines, Karpas 299 and SU-DHL-1. Using cDNA microarrays, ALK(+) ALCL cell lines consistently expressed the highest TIMP1 level among 29 lymphoma cell lines of various subtypes. The association between TIMP1 expression and high level of STAT3 activation was validated by Western blots and immunostaining using antibodies specific for pSTAT3 and TIMP1. We further evaluated the relationship between TIMP1 expression and STAT3 activation in 43 ALCL tumors (19 ALK(+) and 24 ALK(-)) using immunohistochemistry and a tissue microarray. The TIMP1(+) group had a mean of 64% pSTAT3(+) cells as compared to 23% pSTAT3(+) cells in the TIMP1(-) group (P = 0.002). As expected, TIMP1 positivity was higher in the ALK(+) group (15 of 19, 79%) compared with the ALK(-) group (5 of 24, 21%; P = 0.0002) because NPM-ALK restricted to ALK(+) tumors was previously shown to activate STAT3. In conclusion, STAT3 directly contributes to the high level of TIMP1 expression in ALK(+) ALCL, and TIMP1 expression correlates with high level of STAT3 activation in ALCL. TIMP1, as a downstream target of STAT3, may mediate the anti-apoptotic effects of STAT3.

Anaplastic Lymphoma Kinase↗
PubMed2003

IIp45, an insulin-like growth factor binding protein 2 (IGFBP-2) binding protein, antagonizes IGFBP-2 stimulation of glioma cell invasion.

Our previous studies have shown that insulin-like growth factor binding protein 2 (IGFBP-2) is frequently overexpressed in the highly invasive glioblastoma multiforme (GBM). By using a yeast two-hybrid system, we identified a gene, invasion inhibitory protein 45 (IIp45), whose protein product bound to IGFBP-2 through the thyroglobulin-RGD region of the C terminus of IGFBP-2. The IIp45 gene is located on chromosome 1p36 and has nine exons. The IIp45 protein has three SEG (segment of low compositional complexity) domains and an integrin-binding RGD motif. The IIp45 protein was not expressed in some GBMs. Functional studies showed that IIp45 inhibited GBM cell invasion both in vitro and in xenograft model. Gene expression profiling studies showed that IIp45 consistently inhibited the expression of cell invasion-associated genes, such as the transcriptional NFkappaB, and its downstream target gene, intercellular adhesion molecule 1. Thus, we report here the isolation and characterization of a gene, IIp45, whose protein product binds to IGFBP-2 and inhibits glioma cell invasion.

Adult↗
PubMed2003

Insulin-like growth factor binding protein 2 enhances glioblastoma invasion by activating invasion-enhancing genes.

Comparison of gene expressing profiles between gliomas with different grades revealed frequent overexpression of insulin-like growth factor binding protein 2 (IGFBP2) in glioblastoma (GBM), the most advanced stage of glioma. To determine whether IGFBP2 is involved in the proliferative and invasive nature of GBM, we established stable SNB19 GBM cell lines that overexpress IGFBP2. Although there was no marked difference in the cell growth between IGFBP2 overexpressing SNB19(BP2) lines when compared with the control cells, these clones showed significantly increased invasive rates when compared with the parental or vector transfected SNB19 cells. Total RNAs from controls and SNB19(BP2) clones were used for microarray analysis to detect IGFBP2-mediated alterations in gene expression. When compared with parental or vector-transfected control cells, SNB19(BP2) cells consistently showed 3-5-fold increase in the expression of matrix metalloproteinase-2 (MMP-2) as well as other invasion related genes. Increased MMP-2 expression in SNB19(BP2) cells was subsequently confirmed by real time reverse-transcription PCR, Western blotting, and gelatin zymography. Furthermore, consistent with increased MMP-2 expression in SNB19(BP2) cells, transient transfection of a MMP-2 promoter/luciferase reporter also resulted in 3-6-fold higher luciferase activity in SNB19(BP2) cells than in parental or vector-transfected control cells. Finally, tissue microarray analysis of 68 GBM tissue specimens showed a significant correlation between the overexpression of IGFBP2 and elevated MMP-2 expression. Taken together, our data provide evidence that IGFBP2 contributes to glioma progression in part by enhancing MMP-2 gene transcription and in turn tumor cell invasion.

Gene Expression Regulation, Neoplastic↗
PubMed2002

Tumor specific gene expression profiles in human leiomyosarcoma: an evaluation of intratumor heterogeneity.

Leiomyosarcomas are malignant smooth muscle tumors characterized by a spectrum of histopathologic features and clinical behavior. Gene expression profiling of leiomyosarcomas may identify differential gene signatures that may allow for the clinical stratification of the tumors. Typically, surgical specimens from these tumors are large and manifest a variegated macroscopic appearance. Because of their large size at the time of diagnosis, sufficient tissue is available for regional and clonal heterogeneity assessment. However, if the gene expression profiles of samples taken from different locations in the tumors are drastically different, biologic classification on the basis of random sample analysis may not be adequate. Therefore, to assess intertumor and intratumor heterogeneity, the authors performed a gene expression study using leiomyosarcoma specimens from three excised sarcomas from an equal number of different patients. Comparisons between tumor and normal samples from the three patients as well as between carefully mapped peripheral and core specimens from the same tumor (excised from one of the patients), were performed. Analysis of the expression profiles demonstrated minimal intratumor variations compared with intertissue variations, indicating homogeneous tumor specific gene expression profiles. The authors also identified genes that are expressed differentially in tumor and normal tissue.

Biomarkers, Tumor↗
PubMed2002

Effect of autophosphorylation on the catalytic and regulatory properties of protein tyrosine kinase Src.

The function of autophosphorylation in Src family protein tyrosine kinases is not fully understood. In this paper we compared the catalytic and ligand-binding properties of autophosphorylated and nonautophosphorylated (control) Src. The following are the main differences we found. First, while both forms had the same K(m) for ATP-Mg, autophosphorylated Src had significantly higher K(m) values for the phosphate-accepting substrates, polyE(4)Y, and RCM-lysozyme. The autophosphorylated form also had significantly higher V(max) values than the control. The substrate specificity, as measured by V(max)/K(m) ratio, was altered by autophosphorylation and was dependent on the phosphate-accepting substrate. Second, while autophosphorylation did not affect Src activation by free Mg(2+), Zn(2+), which inhibited Src by competing against an essential Mg(2+) activator, inhibited the control threefold more potently than the autophosphorylated form. Third, autophosphorylation significantly reduced the ability of its SH2 domain to bind phosphotyrosine. Fourth, a Pro-rich Src SH3 domain binding peptide activated the control, but not the autophosphorylated Src even though the apparent binding affinity was not significantly affected by autophosphorylation. These differences indicated that autophosphorylation induced significant and widespread changes in the catalytic and regulatory properties of Src. The implications of these findings relative to Src biological regulation are discussed.

CSK Tyrosine-Protein Kinase↗
PubMedDate not supplied

Apoptotic response to 5-fluorouracil treatment is mediated by reduced polyamines, non-autocrine Fas ligand and induced tumor necrosis factor receptor 2.

5-fluorouracil (5-FU) is the major chemotherapeutic agent for treatment of colorectal carcinoma, but the molecular mechanisms of response and resistance are not understood completely. We therefore studied the 5-FU dose response and time course of gene expression transcriptome changes in colon carcinoma cell lines that are relatively sensitive to or resistant to 5-FU (RKO and HT29, respectively. We identified cellular pathways and corroborated functions of selected pathways. Expression of genes for polyamine biosynthesis, i.e., ornithine decarboxylase (ODC) and spermine and spermidine synthases, was repressed in the sensitive line, while the biosynthesis-inhibiting gene ODC antizyme was induced in the resistant line. The rate-limiting gene in catabolism, spermine/spermidine acetyltransferase, was induced in both lines. Polyamine levels showed corresponding drastic decreases after 5-FU treatment, and polyamine replenishment interfered with 5-FU-induced apoptosis. In the sensitive cells which have wild-type p53, the p53 gene and its downstream genes including p21/WAF1, mdm2, Fas, mic-1, EphA2, and ferredoxin reductase as well as genes in the tumor necrosis factor (TNF) pathway including TNF receptor 2 (TNFR2) were induced, but not Fas ligand (FasL). Exposure to exogenous FasL increased 5-FU-induced apoptosis, and anti-TNFR2 antibody, but not anti-TNFR1, partially protected the sensitive cells. Our combination of gene expression profiling and corroborative functional studies revealed that reduced polyamine levels, non-autocrine FasL originating exogenous to tumor cells, and induced TNFR2 are all functional mediators of apoptosis caused by 5-FU in colon carcinoma cells.

Acetyltransferases↗