Member Of RAS Oncogene Family Research Articles

RAN (RAN, member RAS oncogene family)

Review on RAN (RAN, member RAS oncogene family), with data on DNA, on the protein encoded, and where the gene is implicated.

RAP1A (RAP1A, member of RAS oncogene family)

Review on RAP1A (RAP1A, member of RAS oncogene family), with data on DNA, on the protein encoded, and where the gene is implicated.

RAP2A (RAP2A, member of RAS oncogene family)

RAP2A (RAP2A, member of RAS oncogene family)

RAP1B (RAP1B, member of RAS oncogene family)

RAP1B (RAP1B, member of RAS oncogene family)

RAP2B (RAP2B, member of RAS oncogene family)

RAP2B (RAP2B, member of RAS oncogene family)

Gene Alterations of Ovarian Cancer Cells Expressing Estrogen Receptors by Estrogen and Bisphenol A Using Microarray Analysis

Since endocrine disrupting chemicals (EDCs) may interfere with the endocrine system(s) of our body and have an estrogenicity, we evaluated the effect(s) of bisphenol A (BPA) on the transcriptional levels of altered genes in estrogen receptor (ER)-positive BG-1 ovarian cancer cells by microarray and real-time polymerase-chain reaction. In this study, treatment with 17β-estradiol (E2) or BPA increased mRNA levels of E2-responsive genes related to apoptosis, cancer and cell cycle, signal transduction and nucleic acid binding etc. In parallel with their microarray data, the mRNA levels of some altered genes including RAB31_MEMBER RAS ONCOGENE FAMILY (U59877), CYCLIN D1 (X59798), CYCLIN-DEPENDENT KINASE 4 (U37022), IGF-BINDING PROTEIN 4 (U20982), and ANTI-MULLERIAN HORMONE (NM_000479) were significantly induced by E2 or BPA in this cell model. These results indicate that BPA in parallel with E2 induced the transcriptional levels of E2-responsive genes in an estrogen receptor (ER)-positive BG-1 cells. In conclusion, these microarray and real-time polymerase-chain reaction results indicate that BPA, a potential weak estrogen, may have estrogenic effect by regulating E2-responsive genes in ER-positive BG-1 cells and BG-1 cells would be the best in vitro model to detect these estrogenic EDCs.

Proteomic approach with LCMS-IT-TOF identified an increase of Rab33B after transient focal cerebral ischemia in mice

BackgroundSeveral proteins are known to be markedly expressed in the brain during cerebral ischemia; however, the changes in protein profiles within the ischemic brain after an ischemic insult have not been fully elucidated. We studied the changes in the ischemic brain proteome after focal cerebral ischemia, induced by middle cerebral artery occlusion (MCAO) in mice.MethodsLCMS-IT-TOF mass spectrometry was used to detect the changes in ischemic brain protein patterns after MCAO. We evaluated the protein expression detected in the ischemic area, by western blotting and immunohistochemistry.ResultsNine unique proteins were identified from the ischemic area at 10 h after ischemic insult. Among these proteins, we focused on Rab33b, a member of RAS oncogene family and we found that Rab33b was up-regulated in the ischemic striatum and the number of Rab33B-positive cells increased in a time-dependent manner. Rab33B colocalized with Iba-1 positive microglia in the ischemic area.ConclusionThese findings suggest that LCMS-IT-TOF is useful for identifying changes in proteins after cerebral ischemia and that Rab33B is partially related to the pathogenesis of transient cerebral ischemia in mice.

Changes in protein expression after neoadjuvant use of aromatase inhibitors in primary breast cancer: a proteomic approach to search for potential biomarkers to predict response or resistance

Objective: Aromatase inhibitors (AI) have been established as a useful hormonal therapy in hormone receptor-expressing breast carcinoma. However, changes in tumor protein expression after exposure to AIs are not necessarily well understood. These changes may provide insight into how breast carcinomas respond or develop into a state of resistance towards AIs, and lead to the discovery of potential biomarkers to predict treatment responses. Methods: Post-menopausal breast cancer patients were recruited to receive 3 months treatment with neoadjuvant AI. Carcinoma tissues were collected before and after the use of AIs and protein expression profiles were compared using two-dimensional gel electrophoresis. Protein spots with different levels of expression were identified using mass spectrometry. Results: A total of 14 matched pairs of tumor tissues were collected. Both up-regulated and down-regulated proteins were selected and identified as follows: heat shock protein 70 protein 2; Cyclin M3, alpha 1 antichymotrypsin precursor; carbonic anhydrase I, cancer antigen 1; SOBP protein; Rho GDP dissociation inhibitor alpha; glyoxalase I with benzyl glutathione inhibitor; lipid-free human apolipoprotein A-I and RAB4A member RAS oncogene family. Among these proteins, heat shock protein 70 demonstrated the most significant positive correlation with clinical response of the patients. Conclusion: After neoadjuvant use of AI, heat shock protein 70 demonstrated the most consistent phenotypic consistency in both up-regulated and down-regulated protein expression levels among the 14 studied pairs of tumor tissue. Other proteins worthwhile exploring were also identified in this study. These proteins could serve as potential predictors for AI response.

Modification of survival pathway gene expression in human breast cancer cells by tetraiodothyroacetic acid (tetrac)

Tetraiodothyroacetic acid (tetrac) inhibits the cellular actions of thyroid hormone initiated at the hormone receptor on plasma membrane integrin αvβ3. Via interaction with the integrin, tetrac is also capable of inhibiting the angiogenic effects of vascular endothelial growth factor and basic fibroblast growth factor. MDA-MB-231 cells are estrogen receptor-negative human breast cancer cells shown to be responsive to tetrac in terms of decreased cell proliferation. Here we describe actions initiated at the cell surface receptor by unmodified tetrac and nanoparticulate tetrac on a panel of survival pathway genes in estrogen receptor-negative human breast cancer (MDA-MB-231) cells. Nanoparticulate tetrac is excluded from the cell interior. Expression of apoptosis inhibitors XIAP (X-linked inhibitor of apoptosis) and MCL1 (myeloid cell leukemia sequence 1) was downregulated by nanoparticulate tetrac in these breast cancer cells whereas apoptosis-promoting CASP2 and BCL2L14 were upregulated by the nanoparticulate formulation. Unmodified tetrac affected only XIAP expression. Expression of the angiogenesis inhibitor thrombospondin 1 (THBS1) gene was increased by both formulations of tetrac, as was the expression of CBY1, a nuclear inhibitor of catenin activity. The majority of differentially regulated Ras-oncogene family members were downregulated by nanoparticulate tetrac. The latter downregulated expression of epidermal growth factor receptor gene and unmodified tetrac did not. Nanoparticulate tetrac has coherent anti-cancer actions on expression of differentially-regulated genes important to survival of MDA-MB-231 cells.

Designing, optimizing, and implementing high-throughput siRNA genomic screening with glioma cells for the discovery of survival genes and novel drug targets

A major challenge for the treatment of cancers, such as glioblastoma multiforme (GBM), has been resistance to radiation and cancer chemotherapeutics. Short interfering RNA (siRNA) based screening may facilitate the identification of genes and pathways essential for cancer cell survival and could enable a more targeted therapeutic approach for the treatment of GBM. Although the commercial availability of siRNA libraries has expanded greatly, detailed methods for the implementation and analysis of genome-scale screens are largely lacking. To annotate the essential genes and pathways for glioma cell survival, we designed, optimized, and implemented a high-throughput siRNA screen in the highly drug and radiation resistant T98G glioma cell line. We developed a rapid, readily available, and simple strategy to optimize siRNA transfection assays in a 384-well plate format based on immunofluorescence studies and inhibition of the non-essential, endogenous gene lamin A/C. We used these transfection conditions to successfully screen a library of 1056 siRNAs targeting 352 unique human genes in a cell-based one gene per well format to identify the genes essential for glioma cell survival and assess the quality of the screening conditions prior to large-scale screening. After developing and applying a median-based outlier detection algorithm for post-screen analysis, we identified the Ras oncogene family member RAN as an essential gene for glioma cell survival. Successful implementation and analysis of this siRNA screen validates our transfection optimization approach and provides guidance for the rapid development of high-throughput siRNA screens in human glioma cells.

C-peptide is internalised in human endothelial and vascular smooth muscle cells via early endosomes

There is increasing evidence that C-peptide exerts intracellular effects in a variety of cells and could be beneficial in patients with type 1 diabetes. Exactly how C-peptide achieves these effects, however, is unknown. Recent reports showed that C-peptide internalised in the cytoplasm of HEK-293 and Swiss 3T3 cells, where it was not degraded for at least 1 h after uptake. In this study, we investigated the hypothesis that C-peptide is internalised via an endocytic pathway and traffics to classic endocytic organelles, such as endosomes and lysosomes. We studied the internalisation of C-peptide in vascular endothelial and smooth muscle cells, two relevant targets of C-peptide activity, by using Alexa Fluor-labelled C-peptide probes in living cells and immunohistochemistry employing confocal laser-scanning microscopy. To examine trafficking to subcellular compartments, we used fluorescent constructs tagged to RAB5A, member RAS oncogene family (RAB5A) to identify early endosomes, or to lysosomal-associated membrane protein 1 (LAMP1) to identify lysosomes. C-peptide internalised in the cytoplasm of cells within punctate structures identified as early endosomes. Internalisation was clearly detectable after 10 min of incubation and was blocked at 4 degrees C as well as with excess of unlabelled C-peptide. A minor fraction of vesicles, which increased with culture time, co-localised with lysosomes. Uptake of C-peptide was reduced by monodansylcadaverine, a pharmacological compound that blocks clathrin-mediated endocytosis, and by nocodazole, which disrupts microtubule assembly. C-peptide internalises in the cytoplasm of cells by endocytosis, as demonstrated by its localisation in early endosomes. Endosomes might represent a signalling station, through which C-peptide might achieve its cellular effects.

Depletion of T-cell intracellular antigen proteins promotes cell proliferation

The transcriptome of TIA-1/TIAR-depleted cells indicates roles in inflammation, cell-cell signaling, immune suppression, angiogenesis, metabolism and cell proliferation.

Identification of candidate molecular markers of nasopharyngeal carcinoma by microarray analysis of subtracted cDNA libraries constructed by suppression subtractive hybridization

To identify differentially expressed genes and scan candidate molecular markers in nasopharyngeal carcinoma (NPC). We constructed four subtracted cDNA libraries using suppression subtractive hybridization technique, then randomly picked about 1200 colonies from the libraries to construct cDNA microarray and analyzed the gene expression profile in 19 NPCs, three NPC-derived cell lines, and 10 chronic inflammation of nasopharyngeal mucosa tissue samples using the cDNA microarray. We used real-time quantitative reverse transcription polymerase chain reaction and in-situ hybridization techniques to confirm our microarray results. The results showed that there were 37 highly expressed colonies and 68 poorly expressed colonies in NPC. Thirty-two known genes were identified by sequencing 105 differentially expressed colonies in NPC. Palate, lung, and nasal epithelium carcinoma (PLUNC)-associated and homo sapien cell division cycle 37 homolog (Saccharomyces cerevisiae)-like 1 (CDC37L1) genes had a higher frequency than others in the 68 poorly expressed colonies in NPC. The frequency of signal transducer and activator of transcription 5A gene was the highest in the 37 highly expressed colonies in NPC; after that were member RAS oncogene family and secreted protein, acidic, cysteine-rich genes. Real-time quantitative reverse transcription-polymerase chain reaction and in-situ hybridization techniques confirmed that the NPC group had a lower frequency of PLUNC and CDC37L1 expression than the groups of chronic inflammation of nasopharyngeal mucosa (P<0.01). The data suggested that PLUNC and CDC37L1 genes might be the putative molecular markers of NPC. For the first time we found that there was a close relationship between CDC37L1 and NPC.

Cell signaling and transcription factor genes expressed during whole body regeneration in a colonial chordate.

BackgroundThe restoration of adults from fragments of blood vessels in botryllid ascidians (termed whole body regeneration [WBR]) represents an inimitable event in the chordates, which is poorly understood on the mechanistic level.ResultsTo elucidate mechanisms underlying this phenomenon, a subtracted EST library for early WBR stages was previously assembled, revealing 76 putative genes belonging to major signaling pathways, including Notch/Delta, JAK/STAT, protein kinases, nuclear receptors, Ras oncogene family members, G-Protein coupled receptor (GPCR) and transforming growth factor beta (TGF-β) signaling. RT-PCR on selected transcripts documented specific up-regulation in only regenerating fragments, pointing to a broad activation of these signaling pathways at onset of WBR. The followed-up expression pattern of seven representative transcripts from JAK/STAT signaling (Bl-STAT), the Ras oncogene family (Bl-Rap1A, Bl-Rab-33), the protein kinase family (Bl-Mnk), Bl-Cnot, Bl-Slit and Bl-Bax inhibitor, revealed systemic and site specific activations during WBR in a sub-population of circulatory cells.ConclusionWBR in the non-vertebrate chordate Botrylloides leachi is a multifaceted phenomenon, presided by a complex array of cell signaling and transcription factors. Above results, provide a first insight into the whole genome molecular machinery of this unique regeneration process, and reveal the broad participation of cell signaling and transcription factors in the process. While regeneration involves the participation of specific cell populations, WBR signals are systemically expressed at the organism level.

Age and gender related differences in human parotid gland gene expression

Objective The present study evaluated differences in gene expression associated with age and gender in the human parotid gland. Design Parotid gland tissue was analysed using the Affymetrix ® GeneChip ® HGU133plus2.0 array. Results Differential gene expression, defined as a statistically significant difference with a 1.5-fold or greater change, was detected in 787 gene probe sets; 467 (∼59%) showed higher expression in females. Several genes associated with saliva secretion were differentially expressed in male and female parotid glands including vesicle-associated membrane protein 3 VAMP3, synaptosomal-associated protein SNAP23, RAS oncogene family member RAB1A and the syntaxin binding protein STXBP1. Evaluation of gene expression in the youngest and the oldest female subjects revealed that the expression of 228 probe sets were altered during aging; 155 genes were up-regulated in the aged female parotid gland. However, of the genes that were altered during aging, 22 of the 30 probes (73%) classified as being associated with immune responses were down-regulated in the aged parotid gland. A panel of differentially expressed, age- and gender-related genes was selected for validation by quantitative, real-time RT-PCR. Comparable differences in gene expression were detected by both Affymetrix ® array and quantitative, real-time RT-PCR methods. Conclusions Our data suggest that salivary gland function may be adversely affected in the aged population due, at least in part, to the altered regulation of several categories of genes. Moreover, the gender specific differences in gene expression identified in the present study correlate with the previously observed sexual dimorphism in salivary gland function.

BCR expression is decreased in meningiomas showing loss of heterozygosity of 22q within a new minimal deletion region

Neurofibromin 2 ( NF2), located on chromosome arm 22q, has been established as a tumor suppressor gene involved in meningioma pathogenesis. In our study, we investigated 149 meningiomas to determine whether there are additional tumor suppressor genes localized on chromosome 22q, apart from NF2, that might be involved in meningioma pathogenesis. The LOH analysis on chromosome 22q identified two regions of deletion: the first one, which is limited to the NF2 gene locus, and the second one, which is outside this location. The new minimal deletion region (MDR) included the following genes: BCR (breakpoint cluster region), RAB36 (a member of RAS oncogene family), GNAZ [guanine nucleotide binding protein (G protein), α-z polypeptide], and RTDR1 (rhabdoid tumor deletion region gene 1). The expression levels of all these genes, including NF2, were subsequently analyzed by quantitative real-time polymerase chain reaction. We observed a significantly lowered expression level of NF2 in meningiomas with 22q loss of heterozygosity (LOH) within NF2 region compared to the one in meningiomas with 22q retention of heterozygosity (ROH, P<0.05). Similarly, BCR showed a significantly lowered expression in meningiomas with 22q LOH within the new MDR compared to cases with 22q ROH ( P<0.05). Our data, together with the already published information considering BCR function suggest that BCR can be considered as a candidate tumor suppressor gene localized on chromosome 22q which may be involved in meningioma pathogenesis.

Integrated Epigenomic Profiling Reveals Aberrant DNA Hypomethylation in LGL and Demonstrates That a Combination of Genetic and Epigenetic Events Results in Leukemic Evolution in Model of Large Granular Lymphocytic Leukemia.

In addition to genetic alterations, epigenetic events have been recently proposed to play a role in the pathogenesis and progression of various malignancies. We wanted to study the role of both global genetic and epigenetic changes during leukemic progression by combining Whole genome methylation, Gene expression and DNA copy number alteration analyses. We used a cell line model of LGL derived from the same patient at two different stages of disease (Daibata, Int J Cancer . 2004; 108(6):845) for comparison. The first cell line, MOTN1, is IL-2 dependant and was derived during indolent course of LGL, while PLT-2 typifies the aggressive phenotype and is IL-2 independent in vitro. Whole genome methylation was analyzed by the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR; Khulan et al, Genome Res. 2006 Aug;16(8)) that uses differential methylation specific restriction digestion by HpaII and MspI followed by amplification, 2 color labeling and cohybridization to quantitatively determine individual promoter methylation. A whole genome human promoter array (Nimblegen) was used to determine the level of methylation of 25626 gene promoters by calculating HpaII/MspI cut fragment intensity ratio. Gene expression analysis was performed using 37K oligo maskless arrays and high density array comparative genomic hybridization (aCGH) was performed at 6Kb resolution (Nimblegen). aCGH showed significantly higher sensitivity when compared to conventional cytogenetic analysis and revealed that a total of 548 genes were deleted and 635 amplified during leukemic progression. Gene amplification led to significantly increased mean expression of important genes such as CDK6, Fyn kinase, DICER, MAP3K9 and deletion led to decreased gene expression of genes implicated in many cancer pathways by Ingenuity functional analysis. Methylation analysis showed that there was a trend towards hypomethylation with disease progression. The mean change in the HpaII/MspI (HELP) Log (2) ratio was 0.67, where a negative value indicates methylation and positive value indicates hypomethylation. Several genes implicated in hematologic diseases were found to be hypomethylated and overexpressed, including CDK8, IGF binding protein 5, HDAC4 and MAPK14. Methylation and suppression of various genes belonging to the suppressive TGF-β and TNF pathways was observed during leukemic evolution. A comparison of primary LGL samples with normal NK cells and CD4 lymphocytes demonstrated a consistent trend towards global hypomethylation. Significantly, many genes hypomethylated in the cell lines were found to be hypomethylated in 2 primary patient samples of NK and T LGL as well; these included members of RAS oncogene family, and many cell cycle regulators. These results show that interplay of both genetic and epigenetic events may be responsible for transformation of malignancies. For example, in our model we observed that pro-proliferative cyclin dependant protein kinases can be overactivated by both mechanisms, CDK6 by gene amplification and CDK8 by DNA hypomethylation. These findings strongly suggest that progression of LGL from the stable to the aggressive phase is associated with significant epigenetic changes, in addition to cytogenetic alterations.

Molecular mechanisms underlying the tumorigenesis of colorectal adenomas: Correlation to activated K-ras oncogene

Mutations of K-ras gene have been demonstrated in 40-50% of colorectal cancer and large adenoma (>1 cm). This study was intended to clarify the correlation between the existence of K-ras oncogene and the pathological features of colorectal adenomas using our recently developed membrane arrays. Moreover, the downstream genes regulated by K-ras oncogene were explored to serve as potential biomarkers in the early diagnosis and risk assessment of patients with colorectal adenoma. Specimens were collected from 70 patients with colorectal adenoma. The alterations of K-ras oncogene were analyzed by direct sequencing and our constructed membrane arrays, respectively. The results of direct sequencing showed that 21 of 70 samples (30%) had K-ras gene mutations. The most frequently mutated sites included codons 12, 13, 15 and 18. Furthermore, activated K-ras oncogene was identified in 18 of 70 (25.7%) adenoma by membrane arrays. Statistical analyses showed that the membrane array had the accuracy of 90.0%, sensitivity of 88.9%, and specificity of 90.4%. The frequency of the mutational sites of K-ras gene was located as follows: codon 12, 100% (4/4); codon 13, 100% (4/4); codon 15, 75% (6/8); and codon 18, 100% (2/2). The analysis of the correlation between the experimental data and pathological characteristics of adenoma showed that activated K-ras oncogenes were significantly associated with the size, number and histology of adenomas (all P<0.001). Finally, we found the downstream genes activated by K-ras oncogene, including B-cell CLL/lymphoma 2 (BCL2), Homo sapiens H2A histone family, member Z (H2AFZ), Homo sapiens RAP1B, member of RAS oncogene family (RAP1B), Homo sapiens T-box 19 (TBX19), Homo sapiens E2F transcription factor 4, p107/p130-binding (E2F4) and matrix metallopeptidase 1 (MMP1), of which were overexpressed in most of all examined adenomas. These genes were then suggested to have functions involved in cell growth. The preliminary results indicated that the accuracy of membrane arrays was comparable to conventional DNA sequencing in the detection of activated K-ras oncogenes. Therefore, we propose that activated K-ras oncogene in colorectal adenomas may play an important role in the subsequent colorectal carcinogenesis through a group of K-ras-related molecular targets.

Thyroid Hormone Responsive Genes in Cultured Human Fibroblasts

Human skin fibroblasts are readily accessible cells for propagation in culture without transformation that can serve for direct pathophysiology studies in subjects with inherited diseases. We thus examined by quantitative fluorescent cDNA microarray analysis the effect of thyroid hormone (TH) on the expression of more than 15,000 genes in fibroblasts of two normal individuals. Fibroblasts from two subjects with resistance to thyroid hormone (RTH) due to mutations in the TH receptor-beta gene were used to confirm the specificity of the hormonal effect by the ability to discriminate between normal cells and cells with a defect in TH action. Microarray analysis identified 148 genes induced by 1.4-fold or more and five genes repressed to 0.7 or less 24 h after treatment with 2 x 10(-9) M T(3). Taking into account duplicate genes, these represented 91 up-regulated and five down-regulated genes, respectively. Confirmation by real-time PCR was obtained in eight of 10 induced and two of three repressed genes that were tested. Further evidence for T(3)-specific induction was provided by a graded dose response absent in fibroblasts from the patients with RTH. The following genes not previously known to be induced by TH were identified and validated: aldo-keto reductase family 1 C1-3, collagen type VI alpha 3, member RAS oncogene family brain antigen RAB3B, platelet phosphofructokinase, hypoxia-inducible factor-1 alpha, and enolase 1 alpha. These genes as well as three known to be TH regulated in other species and found in this study also in human cells (glucose transporter 1, solute carrier family 16 member 3, and basic transcription element-binding protein 1) have a variety of regulatory functions in development and metabolism. TH seems to induce these genes by initiating either genomic or nongenomic mechanisms. Surprisingly, TH-mediated down-regulation of fibroblast growth factor 7 and alcohol dehydrogenase 1B persisted in fibroblasts from patients with RTH. This first systematic study of TH-mediated gene expression in normal human cells identifies several new TH-responsive genes and demonstrates that skin fibroblasts are suitable for the study of TH action in health and disease.

Gene expression profile changes following erlotinib treatment in patients with metastatic breast cancer

577 Background: We previously described changes in EGFR kinase activity and downstream signaling pathways using IHC in tumor biopsies in 15 patients with metastatic breast cancer pre and post treatment with erlotinib (150 mg daily for 1 month). There were no clinical responses and no changes in Ki67 in tumors after erlotinib. One tumor expressed significant levels of EGFR which decreased after treatment. Aims of this study are to delineate erlotinib induced changes in gene expression profiles in EGFR positive (+) and negative (-) tumors. Methods: RNA was isolated from frozen tumor cores and gene expression profiles in 9 paired pre-post treatment tumors were measured with Affymetrix U133A GeneChip containing 22283 human genes. Results were analyzed using Affymetrix MS5.0 comparison analysis algorithm and principle components analysis. EGFR expression was validated by IHC. Results: Only 1/9 tumor expressed EGFR by IHC. cDNA array data were 100% concordant with IHC for EGFR expression. In EGFR + tumor, EGFR RNA did not change with erlotinib treatment. About 300 genes were significantly changed in distinct paired tumors, independent of EGFR expression. These included genes encoding extracellular matrix components, matrix metalloproteinases, drug metabolism, protein translation, and transcription factors. EGFR interacting genes, tissue inhibitor of metalloproteinases-1 and -3 were up-regulated > 2 fold in the EGFR+ tumor, down-regulated > 2 fold in 6 EGFR- tumors, unchanged in 2 EGFR- tumors. Examples of other altered genes are: Rab GTPases - members of ras oncogene family; heterogeneous nuclear ribonucleoprotein C (C1/C2); insulin-like growth factor binding protein 3, 4, 5, 7; serine proteinase inhibitor, clade A; heat shock 70kDa protein 1B; cytochrome P450 family members. Conclusions: Erlotinib is associated with changes in gene expression in EGFR+ and –tumors. This suggests that the drug has additional targets. The decrease in EGFR protein induced by erlotinib in the EGFR+ tumor was not due to transcriptional effects and may be due to the accelerated ubiquitylation and degradation of the receptor. Validation by IHC and real-time RT-PCR is in progress. No significant financial relationships to disclose.