Showing papers by "Thomas D. Schmittgen published in 2006"

Expression profiling identifies microRNA signature in pancreatic cancer

Abstract: microRNAs are functional, 22 nt, noncoding RNAs that negatively regulate gene expression. Disturbance of microRNA expression may play a role in the initiation and progression of certain diseases. A microRNA expression signature has been identified that is associated with pancreatic cancer. This has been accomplished with the application of real-time PCR profiling of over 200 microRNA precursors on specimens of human pancreatic adenocarcinoma, paired benign tissue, normal pancreas, chronic pancreatitis and nine pancreatic cancer cell lines. Hierarchical clustering was able to distinguish tumor from normal pancreas, pancreatitis and cell lines. The PAM algorithm correctly classified 28 of 28 tumors, 6 of 6 normal pancreas and 11 of 15 adjacent benign tissues. One hundred microRNA precursors were aberrantly expressed in pancreatic cancer or desmoplasia (p < 0.01), including microRNAs previously reported as differentially expressed in other human cancers (miR-155, miR-21, miR-221 and miR-222) as well as those not previously reported in cancer (miR-376a and miR-301). Most of the top aberrantly expressed miRNAs displayed increased expression in the tumor. Expression of the active, mature microRNA was validated using a real-time PCR assay to quantify the mature microRNA and Northern blotting. Reverse transcription in situ PCR showed that three of the top differentially expressed miRNAs (miR-221, -376a and -301) were localized to tumor cells and not to stroma or normal acini or ducts. Aberrant microRNA expression may offer new clues to pancreatic tumorigenesis and may provide diagnostic biomarkers for pancreatic adenocarcinoma.

Increased expression of microRNA-155 in Epstein-Barr virus transformed lymphoblastoid cell lines

Abstract: In a recent issue of Genes, Chromosomes & Cancer, Metzler et al. identified that the precursor to microRNA-155 (miR-155) was located within exon 3 of the noncoding RNA BIC (Metzler et al., 2004). In a prior issue of Genes, Chromosomes & Cancer, BIC was reported to be expressed at very high levels in Hodgkin lymphoma cell lines compared to nonHodgkin lymphoma cell lines (van den Berg et al., 2003). Increased BIC RNA was found in the lymphoblastoid cell lines JY25 and CB33 (Eis et al., 2005) and to a lesser extent in POP and RAY (van den Berg et al., 2003). The expression of miR-155 precursor was increased by 100-fold in children with Burkitt lymphoma compared to normal blood and pediatric leukemia patients (Metzler et al., 2004). Increased mature miR-155 was reported in cell lines and patient specimens of diffuse large Bcell lymphoma (DCBCL) and Hodgkin lymphoma (Eis et al., 2005). BIC was originally identified as a transcriptionally activated gene by promoter insertion at a common retroviral integration site in B-cell lymphomas induced by the avian leukemia virus (Tam et al., 1997). Identification of miR-155 within the BIC gene (Metzler et al., 2004) and subsequent expression of mature miR-155 in patient samples and cell lines (Eis et al., 2005) strengthens the case that the functional oncogenic product of BIC is the 21 nucleotide mature miR-155. This is supported by other evidence including that the miR-155 precursor is located within the highly conserved region of BIC (Tam, 2001). Differential miRNA expression has been shown in a number of human cancers (Calin et al., 2002; Michael et al., 2003; Takamizawa et al., 2004; Johnson et al., 2005); however the expression of most of these miRNAs are downregulated. miR-155 is one of the few miRNAs whose expression is increased in cancer. miRNAs are believed to function by binding to the 30 untranslated region of protein coding mRNAs by partial complimentarity, resulting in reduced translation of the target gene. We have recently developed a high throughput, real-time PCR assay to profile the expression of human miRNA precursors (Schmittgen et al., 2004; Jiang et al., 2005). The assay was used to profile the expression of 222 miRNA precursors in CD19þ B cells from three donors and in the lymphoblastoid cell line Wa C3 CD5þ. Wa C3 CD5þ was derived by immortalizing peripheral blood lymphocytes from a B-CLL patient by using Epstein-Barr virus, S. aureus Cowan 1, MP6-thioredoxin, and interleukin-2 (Wendel-Hansen et al., 1994). The expression of most of the miRNA precursors was comparable in CD19þ B cells and Wa C3 CD5þ (Fig. 1). The expression of miR-155 precursor was markedly up-regulated in Wa C3 CD5þ, representing a 110-fold higher expression in Wa C3 CD5þ compared to CD19þ B cells (Fig. 1). We were intrigued by the study of Küppers et al. (2003), who performed gene expression array analysis on various hematopoietic cancer cell lines including Hodgkin lymphoma, DLBCL, (GC and ABC types), lymphoblastoid cell lines, and Burkitt lymphoma (Küppers et al., 2003). The data were clustered into two groups based upon similarities to the expression profile of the ABC or GC phenotypes. The ABC-like cluster contained Hodgkin lymphoma (L-428, L-1236, KMH2, and HDLM2), DLBCL (Ly3 and Ly10), and EBV-transformed lymphoblastoid cell lines (Daikiki, NC6, CB33, RD, and IARC304) (Küppers et al., 2003). The GC cluster contained twelve cell lines including GC DLBCL (Ly1, Ly7, and Val) and Burkitt lymphoma lines Ramos, EB3, and Namalwa (Küppers et al., 2003). Interestingly, all of the cell lines with known miR-155 overexpression (L-428, L-1236, KM-H2, HDLM-2, Ly3, and Ly10 (Eis et al., 2005)) were present only in the ABC-like cluster, while those with basal miR-155 expression (Ramos, Ly8 and Ly1 (Eis et al., 2005)) were present in the GC cluster (Küppers et al., 2003). Since miR-155 was increased in Wa C3 CD5þ cells (Fig. 1) and BIC was increased in JY25 and CB33 (Eis et al., 2005), we hypothesized that miR-155 may be overexpressed in the EBV trans-

Apoptosis induced by novel aldehyde calpain inhibitors in human tumor cell lines

Abstract: Calpain is a class of Ca(2+)-dependent cysteine proteases and has been suggested to be involved in several important signaling cascades. A series of novel aldehyde calpain inhibitors identified in our laboratory were more potent and specific than commercially available calpain inhibitors, and were used to assess the involvement of calpain in cancer. Our inhibitors demonstrated potent anti-proliferative activity in four cancer cell lines (PC-3, HeLa, Jurkat and Daudi) with IC(50)'s ranging from 2 to >30 microM. A non-cancer cell line (CV-1) was 4-7-fold less sensitive than the cancer cell lines. Apoptotic activity was determined and appeared to be inversely correlated to calpain expression levels in the different cell types. Leukemia cell lines (i.e., Daudi and Jurkat) with undetectable m-calpain were more susceptible to the apoptotic effects in response to calpain inhibition, while apoptosis was not detected in PC-3 prostate cancer cells, which highly express m-calpain. The extent of apoptosis in HeLa cells was moderate under identical conditions. Apoptosis induced by calpain inhibition was accompanied by caspase-3 activation. Furthermore, cell cycle analysis showed that aldehyde calpain inhibitors arrested cells at the G2/M boundary in a concentration-dependent manner. These results indicate that aldehyde calpain inhibitors exhibit their cytotoxic effects via induction of G2/M arrest and apoptosis. Importantly, the compounds failed to exert any inhibitory effects toward 20S proteasome. Collectively, our results suggest that calpain is a novel target for the treatment of a variety of cancer diseases and provide leads for further discovery and development of calpain inhibitors.

Methods for quantifying microRNA precursors

Abstract: The present invention is directed to methods, reagents, kits and compositions for detecting microRNA (miRNA) precursors in a biological sample. The method uses gene-specific primers and reverse transcriptase to convert the primary miRNA precursors (pri-miRNA) and pre-miRNA precursors (pre-miRNAs) to cDNA. The method also uses amplification reactions using gene specific forward and reverse primers that are targeted to the hairpin sequence of pri- and pre-microRNA precursors to detect the expression levels of both the pri- and the pre-micoRNAs.