Lu Charboneau

Reverse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front.

Protein arrays are described for screening of molecular markers and pathway targets in patient matched human tissue during disease progression. In contrast to previous protein arrays that immobilize the probe, our reverse phase protein array immobilizes the whole repertoire of patient proteins that represent the state of individual tissue cell populations undergoing disease transitions. A high degree of sensitivity, precision and linearity was achieved, making it possible to quantify the phosphorylated status of signal proteins in human tissue cell subpopulations. Using this novel protein microarray we have longitudinally analysed the state of pro-survival checkpoint proteins at the microscopic transition stage from patient matched histologically normal prostate epithelium to prostate intraepithelial neoplasia (PIN) and then to invasive prostate cancer. Cancer progression was associated with increased phosphorylation of Akt (P<0.04), suppression of apoptosis pathways (P<0.03), as well as decreased phosphorylation of ERK (P<0.01). At the transition from histologically normal epithelium to PIN we observed a statistically significant surge in phosphorylated Akt (P<0.03) and a concomitant suppression of downstream apoptosis pathways which proceeds the transition into invasive carcinoma.

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

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.

Sensitive Immunoassay of Tissue Cell Proteins Procured by Laser Capture Microdissection

Coupling laser capture microdissection (LCM) with sensitive quantitative chemiluminescent immunoassays has broad applicability in the field of proteomics applied to normal, diseased, or genetically modified tissue. Quantitation of the number of prostate-specific antigen (PSA) molecules/cell was conducted on human prostate tissue cells procured by LCM from fixed and stained frozen sections. Under direct microscopic visualization, laser shots 30 microm in diameter captured specific cells from the heterogeneous tissue section onto a polymer transfer surface. The cellular macromolecules from the captured cells were solubilized in a microvolume of extraction buffer and directly assayed using an automated (1.5 hour) sandwich chemiluminescent immunoassay. Calibration of the chemiluminescent assay was conducted by developing a standard curve using known concentrations of PSA. After the sensitivity, precision, and linearity of the chemiluminescent assay was verified for known numbers of solubilized microdissected tissue cells, it was then possible to calculate the number of PSA molecules per microdissected tissue cell for case samples. In a study set of 20 cases, using 10 replicate samples of 100 laser shots per sample, the within-run (intraassay) SD was approximately 10% of the mean or less for all cases. In this series the number of PSA molecules per microdissected tissue cell ranged from 2 x 10(4) to 6. 3 x 10(6) in normal epithelium, prostate intraepithelial neoplasia (PIN), and invasive carcinoma. Immunohistochemical staining of human prostate for PSA was compared with the results of the soluble immunoassay for the same prostate tissue section. Independent qualitative scoring of anti-PSA immunohistochemical staining intensity paralleled the LCM quantitative immunoassay for each tissue subpopulation and verified the heterogeneity of PSA content between tissue subpopulations in the same case. Extraction buffers were successfully adapted for both secreted and membrane-bound proteins. This technology has broad applicability for the quantitation of protein molecules in pure populations of tissue cells.

Similarities of prosurvival signals in Bcl-2-positive and Bcl-2-negative follicular lymphomas identified by reverse phase protein microarray

Overexpression of Bcl-2 protein has been known to play a role in the pathogenesis of follicular lymphoma (FL). However, 10–15% of FLs are negative for Bcl-2 by immunohistochemistry, raising the possibility that another gene product(s) may provide prosurvival signal(s). We used reverse phase protein microarray to analyze lysates of follicle center cells isolated by laser capture microdissection from: Bcl-2+ FL, Bcl-2− FL and reactive follicular hyperplasia (FH) (nine cases each group). TUNEL assay confirmed similar and reduced levels of apoptosis in Bcl-2+ FL and Bcl-2− FL, indicating the likelihood of Bcl-2-independent inhibition of apoptosis. Arrays were quantitatively analyzed with antibodies to proteins involved in the apoptotic pathway. As expected, Bcl-2 levels were up to eight-fold higher in Bcl-2+ FL than in FH and Bcl-2− FL. However, there was no difference in levels of Mcl-1 and survivin among these three groups. Bcl-XL showed a trend for increased expression in Bcl-2− FL as compared with Bcl-2+ FL, although the differences did not reach statistical significance (P>0.1). The increase in Bcl-XL may provide an alternative antiapoptotic signal in FL negative for Bcl-2 protein. Interestingly, Bax expression was higher in FL (Bcl-2+ or −) than in FH (P=0.001). Notably, phospho-Akt (Ser-473) was increased in FL (Bcl-2+ or −) (P<0.03) with increased phospho-Bad (Ser-136), as compared with levels in FH. The activation of the Akt/Bad pathway provides further evidence of prosurvival signals in FL, independent of Bcl-2 alone. These data suggest that nodal FL represents a single disease with a final common biochemical pathway.

Laser Capture Microdissection: Beyond Functional Genomics to Proteomics

Proteomics will drive biology and medicine beyond genomics, and can have a profound impact on molecular diagnostics. The posttranslational modifications of cellular proteins that govern physiology and become deranged in disease cannot be accurately portrayed by gene expression alone. Consequently, new technology is being developed to discover, and quantitatively monitor, proteomic changes that are associated with disease etiology and progression. In the past, proteomic technologies were restricted to tumor cell lines or homogenized bulk tissue specimens. This source material may not accurately reflect molecular events taking place in the specific cells of the tissue itself. This article describes a completely new class of proteomic-based approaches aimed at the identification and investigation of protein markers in the actual histologically defined cell populations that are immersed in heterogeneous diseased tissue. It is envisioned that these investigations will eventually lead to novel diagnostic, prognostic, or therapeutic markers that can be applied to monitor therapeutic toxicity or efficacy.

APPLICATION OF LASER CAPTURE MICRODISSECTION AND PROTEIN MICROARRAY TECHNOLOGIES IN THE MOLECULAR ANALYSIS OF AIRWAY INJURY FOLLOWING POLLUTION PARTICLE EXPOSURE

Understanding the mechanisms by which various types of air pollution particles (particulate matter, PM) mediate adverse health effects would provide biological plausibility to epidemiological associations of increased rates of morbidity and mortality. The majority of information regarding the means by which PM generates lung injury has been derived from in vitro studies. However, it is unclear as to what extent these mechanisms can be extrapolated to the in vivo situation. Current methods to assess mechanisms of PM-induced lung injury make it difficult to obtain site-specific, sensitive, and comprehensive determinations of cellular and molecular pathology associated with PM-induced injury. In the present study, the ability of laser capture microdissection (LCM) and protein microarray technologies were assessed to examine the effect of residual oil fly ash (ROFA) exposure on airway intracellular signaling pathways and transcription factor activation. Sprague-Dawley rats were intratracheally instilled with 0.5 mg/rat of ROFA. LCM was used to recover airway cells and protein extracts derived from the microdissected airways were analyzed by protein microarray. ROFA exposure increased p-ERK:ERK and p-IκB:IκB, suggesting changes in cell growth, transformation, and inflammation within the airway. These results are consistent with previously reported in vitro findings, demonstrating for the first time the credibility of applying LCM and protein microarray technologies to assess acute lung injury induced by environmental air pollutants.

Laser Capture Microdissection

This unit describes laser capture microdissection (LCM) using the Pixcell II as a technique to provide the scientific community with the opportunity to perform molecular analyses on pure cell populations procured directly from tissues. After identifying specific cells of interest, the cells are captured by firing a near infrared laser through a thermaplastic polymer film that rests on top of the cells. The cells are then ready for molecular analyses.

Overview of metastasis assays.

During tumor progression, cells acquire genetic and proteomic changes as they transform from normal to hyperplastic, through dysplasia, to carcinoma in situ, and finally to invasive and metastatic. The time course of progression may extend as far back as 10 years prior to diagnosis. Discerning the mechanism whereby tumor cells execute metastatic dissemination may provide the foundation necessary for successful treatment of the disease. For example, direct genetic evidence has linked in situ breast cancer to invasive carcinoma of the breast supporting the generally accepted assumption that carcinoma in situ of the breast is a clonal expansion of hyperproliferating cells. This in turn may provide a more comprehensive and/or functionally directed target strategy for intervention and prevention of breast cancer. This overview provides a picture of the processes related to metastasis and the experimental approaches used to study these processes.

Micro RT-PCR.

This unit presents a collection of protocols for the microisolation, manipulation, and amplification of the RNA content of microdissected cells. Even though emphasis in these protocols is given for microdissected cells, these protocols have successfully been used for bulk tissue (i.e., less than 10 ug).