Proteomic profiling of the NCI-60 cancer cell lines using new high-density reverse-phase lysate microarrays

Abstract

Because most potential molecular markers and targets are proteins, proteomic profiling is expected to yield more direct answers to functional and pharmacological questions than does transcriptional profiling. To aid in such studies, we have developed a protocol for making reverse-phase protein lysate microarrays with larger numbers of spots than previously feasible. Our first application of these arrays was to profiling of the 60 human cancer cell lines (NCI-60) used by the National Cancer Institute to screen compounds for anticancer activity. Each glass slide microarray included 648 lysate spots representing the NCI-60 cell lines plus controls, each at 10 two-fold serial dilutions to provide a wide dynamic range. Mouse monoclonal antibodies and the catalyzed signal amplification system were used for immunoquantitation. The signal levels from the >30,000 data points for our first 52 antibodies were analyzed by using P-SCAN and a quantitative dose interpolation method. Clustered image maps revealed biologically interpretable patterns of protein expression. Among the principal early findings from these arrays were two promising pathological markers for distinguishing colon from ovarian adenocarcinomas. When we compared the patterns of protein expression with those we had obtained for the same genes at the mRNA level by using both cDNA and oligonucleotide arrays, a striking regularity appeared: cell-structure-related proteins almost invariably showed a high correlation between mRNA and protein levels across the NCI-60 cell lines, whereas non-cell-structure-related proteins showed poor correlation.

Detailed DNA methylation profiles of the E-cadherin promoter in the NCI-60 cancer cells

“…However, we have found that the two cell lines are extraordinarily similar to the five NCI-60 melanotic melanomas in their profiles of sensitivity to thousands of drugs in the NCI screen (36), their transcript expression profiles (as assessed using six different microarray and RT-PCR platforms; refs. 36, 47 -51), and their protein expression profiles as assessed using two-dimensional gels (34) and reverse-phase lysate arrays (52,53). Independent evidence supporting melanocytic origin has now been presented by others (54).…”

Challenges in cancer research and multifaceted approaches for cancer biomarker quest

“…The specificity of each of the antibodies was verified by Western blotting. The NCI-60 panel contains 2-9 cell lines per each histological tumor type including leukemias, melanomas, and carcinomas of ovaries, renal, breast, prostate, colon, lung and central nervous system [75]. The drug activities of 118 agents were measured by growth inhibition within 48 h. In order to establish the classifiers that would be independent of tissue of origin, a combination of several algorithms were used.…”

Quantum dots-based reverse phase protein microarray

“…The DNA-PKcs protein was kindly provided by Dr. Scott R. Peterson and prepared as described before [30]. For protein lysate microarrays, the cultured cells were collected by scraping, and protein lysates were prepared from the cells according to the procedure of Nishizuka et al [31] Briefly, cells were …”