Identical probes on different high-density oligonucleotide microarrays can produce different measurements of gene expression

BACKGROUND: There are many potential sources of variability in a microarray experiment. Variation can arise from many aspects of the collection and processing of samples for gene expression analysis. Oligonucleotide-based arrays are thought to minimize one source of variability as identical oligonucleotides are expected to recognize the same transcripts during hybridization. RESULTS: We demonstrate that although the probes on the U133A GeneChip arrays are identical in sequence to probes designed for the U133 Plus 2.0 arrays the values obtained from an experimental hybridization can be quite different. Nearly half of the probesets in common between the two array types can produce slightly different values from the same sample. Nearly 70% of the individual probes in these probesets produced array specific differences. CONCLUSION: The context of the probe may also contribute some bias to the final measured value of gene expression. At a minimum, this should add an extra level of caution when considering the direct comparison of experiments performed in two microarray formats. More importantly, this suggests that it may not be possible to know which value is the most accurate representation of a biological sample when comparing two formats

The gene expression profiles of primary and metastatic melanoma yields a transition point of tumor progression and metastasis

<p>Abstract</p> <p>Background</p> <p>The process of malignant transformation, progression and metastasis of melanoma is poorly understood. Gene expression profiling of human cancer has allowed for a unique insight into the genes that are involved in these processes. Thus, we have attempted to utilize this approach through the analysis of a series of primary, non-metastatic cutaneous tumors and metastatic melanoma samples.</p> <p>Methods</p> <p>We have utilized gene microarray analysis and a variety of molecular techniques to compare 40 metastatic melanoma (MM) samples, composed of 22 bulky, macroscopic (replaced) lymph node metastases, 16 subcutaneous and 2 distant metastases (adrenal and brain), to 42 primary cutaneous cancers, comprised of 16 melanoma, 11 squamous cell, 15 basal cell skin cancers. A Human Genome U133 Plus 2.0 array from Affymetrix, Inc. was utilized for each sample. A variety of statistical software, including the Affymetrix MAS 5.0 analysis software, was utilized to compare primary cancers to metastatic melanomas. Separate analyses were performed to directly compare only primary melanoma to metastatic melanoma samples. The expression levels of putative oncogenes and tumor suppressor genes were analyzed by semi- and real-time quantitative RT-PCR (qPCR) and Western blot analysis was performed on select genes.</p> <p>Results</p> <p>We find that primary basal cell carcinomas, squamous cell carcinomas and thin melanomas express dramatically higher levels of many genes, including <it>SPRR1A/B</it>, <it>KRT16/17</it>, <it>CD24</it>, <it>LOR</it>, <it>GATA3</it>, <it>MUC15</it>, and <it>TMPRSS4</it>, than metastatic melanoma. In contrast, the metastatic melanomas express higher levels of genes such as <it>MAGE</it>, <it>GPR19</it>, <it>BCL2A1</it>, <it>MMP14</it>, <it>SOX5</it>, <it>BUB1</it>, <it>RGS20</it>, and more. The transition from non-metastatic expression levels to metastatic expression levels occurs as melanoma tumors thicken. We further evaluated primary melanomas of varying Breslow's tumor thickness to determine that the transition in expression occurs at different thicknesses for different genes suggesting that the "transition zone" represents a critical time for the emergence of the metastatic phenotype. Several putative tumor oncogenes (<it>SPP-1</it>, <it>MITF</it>, <it>CITED-1</it>, <it>GDF-15</it>, <it>c-Met</it>, <it>HOX </it>loci) and suppressor genes (<it>PITX-1</it>, <it>CST-6</it>, <it>PDGFRL</it>, <it>DSC-3</it>, <it>POU2F3</it>, <it>CLCA2</it>, <it>ST7L</it>), were identified and validated by quantitative PCR as changing expression during this transition period. These are strong candidates for genes involved in the progression or suppression of the metastatic phenotype.</p> <p>Conclusion</p> <p>The gene expression profiling of primary, non-metastatic cutaneous tumors and metastatic melanoma has resulted in the identification of several genes that may be centrally involved in the progression and metastatic potential of melanoma. This has very important implications as we continue to develop an improved understanding of the metastatic process, allowing us to identify specific genes for prognostic markers and possibly for targeted therapeutic approaches.</p

Delta-Opioid Receptor (δOR) Targeted Near-Infrared Fluorescent Agent for Imaging of Lung Cancer: Synthesis and Evaluation In Vitro and In Vivo

In the United States, lung cancer is the leading cause of cancer death and ranks second in the number of new cases annually among all types of cancers. Better methods or tools for diagnosing and treating this disease are needed to improve patient outcomes. The delta-opioid receptor (δOR) is reported to be overexpressed in lung cancers and not expressed in normal lung. Thus, we decided to develop a lung cancer-specific imaging agent targeting this receptor. We have previously developed a δOR-targeted fluorescent imaging agent based on a synthetic peptide antagonist (Dmt-Tic) conjugated to a Cy5 fluorescent dye. In this work, we describe the synthesis of Dmt-Tic conjugated to a longer wavelength near-infrared fluorescent (NIRF) dye, Li-cor IR800CW. Binding affinity of Dmt-Tic-IR800 for the δOR was studied using lanthanide time-resolved fluorescence (LTRF) competitive binding assays in cells engineered to overexpress the δOR. In addition, we identified lung cancer cell lines with high and low endogenous expression of the δOR. We confirmed protein expression in these cell lines using confocal fluorescence microscopy imaging and used this technique to estimate the cell-surface receptor number in the endogenously expressing lung cancer cell lines. The selectivity of Dmt-Tic-IR800 for imaging of the δOR in vivo was shown using both engineered cell lines and endogenously expressing lung cancer cells in subcutaneous xenograft models in mice. In conclusion, the δOR-specific fluorescent probe developed in this study displays excellent potential for imaging of lung cancer

Characterization of a R115777 Resistant Human Multiple Myeloma Cell Line with Cross-Resistance to PS-341

Abstract Multiple myeloma is an incurable disease where new treatment strategies are required to improve on current treatment standards. Based on this knowledge we have performed a phase II clinical trial testing the farnesyltransferase inhibitor R115777 in patients with relapsed myeloma (Alsina M. et al, Blood 2004). R115777 was found to have a favorable toxicity profile and 64% of patients achieved disease stabilization. RAS mutation and inhibition of farnesyltransferase did not correlate with clinical efficacy; findings consistent with our prior observation that R115777 induces apoptosis via a Ras independent mechanism (Beaupre D.M. et al, Mol. Cancer Ther. 2004). In order to further characterize the mechanisms by which R115777 induces apoptosis in myeloma cells and to investigate drug resistance, we have identified and characterized a R115777 resistant human myeloma cell line. 8226/S cells were cultured continuously in increasing concentrations of R115777 for over 6 months. 8226/R5 cells were found to be nearly 50 times more resistant to R115777 compared to the parent cell line. K-Ras remained prenylated in both resistant and sensitive cells after R115777 treatment; however, HDJ-2 farnesylation was inhibited in both lines implying that farnesyltranseferase (the drug target) is not modified. 8226/R5 cells were also resistant to a diverse group of anti-tumor agents including PS-341. Many 8226 lines that acquire drug resistance have elevated expression of P-glycoprotein. We found that P-glycoprotein expression is not increased in the 8226/R5 line and furthermore influx and efflux of R115777 was similar in both parent and resistant cells. Expression of the drug resistance proteins hsp27, 70, and 90 were also not increased. Comparison of 8226/S and 8226/R5 gene expression profiles revealed increased expression of JAK2 in resistant cells. Interestingly, STAT3 phosphorylation was significantly increased in the 8226/R5 line consistent with its reported role in myeloma drug resistance. Experiments are underway to delineate the contribution of JAK2 to the multidrug resistant phenotype. Characterization of aberrant JAK2 activation is relevant since constitutive STAT3 activity is frequently observed in primary myeloma isolates