Deleted in liver cancer 3 (DLC-3), a novel Rho GTPase-activating protein, is downregulated in cancer and inhibits tumor cell growth.

Two related Rho GTPase-activating proteins, DLC-1 (deleted in liver cancer 1) and DLC-2, are emerging as bona fide tumor suppressor genes that inhibit cancer cell growth. In this report, we characterized a gene on chromosome Xq13 that encodes DLC-3 (also known as KIAA0189 and STARD8), a third member of the DLC family. The DLC-3 gene has transcripts with alternative 5' ends, one of which, DLC-3alpha, encodes an 1103-amino acid polypeptide highly similar to DLC-1 and DLC-2. A second isoform (DLC-3beta) would yield a protein lacking the N-terminal sterile alpha motif domain. The DLC-3 gene is widely expressed in normal tissues, but DLC-3 mRNA levels were low or absent in a significant number of breast, ovarian, liver and prostate cancer cell lines. Using a cancer profiling array to compare matched tumor and normal human tissues, downregulation of DLC-3 mRNA was observed in kidney, lung, ovarian, uterine and breast cancer samples. By quantitative reverse transcriptase-polymerase chain reaction, DLC-3 expression was reduced in primary prostate carcinomas relative to normal prostate tissue. Transfection of human breast and prostate cancer cells with a DLC-3alpha expression vector inhibited cell proliferation, colony formation and growth in soft agar. These results indicate that deregulation of DLC-3 may contribute to breast and prostate tumorigenesis.

Gene expression during camptothecin-induced apoptosis in human myeloid leukemia cell line ML-2.

Malignant cell proliferation and accumulation depends on the balance between the rates of cell production and cell death. Recent evidence indicates that apoptosis is important in the development of cancer. Apoptosis is strictly controlled by various regulators, which can take part in the apoptotic process, proliferation and differentiation alike. Apoptosis was induced in myeloid cell line ML-2 by camptothecin, an inhibitor of topoisomerase I. After 18 hours of induction by camptothecin 50% of cells were apoptotic. The apoptotic effect of CAM was reversible in the cells studied. The induction of apoptosis influenced the expression of apoptosis and cell cycle regulators as detected by cDNA arrays, RT-PCR or Western blotting. According to cDNA arrays e.g. bax, bfl1, bak, pRb2, c-jun, jun-B were upregulated, and cdk4, cyclin B1, wee1, CRAF1, DP1 were downregulated. A number of other regulators like p21 and cdc25A, as well as some other genes linked with apoptosis, as p53 and the bcl-2 family, were up- or down-regulated as determined by real-time PCR. Changes in gene expression were found not only in the group of regulators of apoptosis and the cell cycle, but also among regulators of differentiation.

The use of housekeeping genes (HKG) as an internal control for the detection of gene expression by quantitative real-time RT-PCR.

Quantitative real-time RT-PCR is a very useful technique for estimating gene expression at the mRNA level. The expression of a tested gene has to be compared with that of a control gene. Various housekeeping genes have been used as control genes in different systems. In our study we tested several housekeeping genes in the model of gene expression after induction of apoptosis and differentiation. The myeloid cell lines were incubated with phorbol esters, butyric acid and combination of TNFalpha and IFNgamma to induce differentiation. Camptothecin was used for induction of apoptosis. Tested control genes included beta2-microglobulin, GAPDH, 18S ribosomal RNA and abl. GAPDH was found to be the best control gene in the apoptotic system. Different control genes were suitable for different systems where differentiation or senescence was induced. Our results show that attention should be paid to the choice of an appropriate control gene of quantitative real-time RT-PCR for different experimental models and various experimental conditions.

Optimized multiplex IgH/ras PCR: a tool for quantitative monitoring of B-lymphoproliferative disorders.

The use of quantitative PCR is recommended to monitor the level of residual hematological malignancies. The proposed multiplex IgH/ras PCR uses a co-amplification of the clonal CDR3 rearrangement of the immunoglobulin heavy chain gene (IgH) as a disease marker and a segment of the Hras 1 gene containing codon 61 (ras) as a control gene. Serial dilutions of stored diagnostic DNAs are examined together in the same PCR at a sub-plateau phase and, after analysis by densitometry, the amount of CDR3 product is related to the ras product. An increase of this ratio at comparable amounts of DNA is viewed as an increase of malignant cells. This endpoint PCR quantifying approach appears to be applicable in monitoring B-lymphoproliferative disorders as was shown to be true in B-cell non-Hodgkin's lymphoma and may provide information on disease activity and treatment outcome.

Monitoring of residual disease in non-Hodgkin's lymphomas by quantitative PCR (preliminary report).

Highly sensitive PCR techniques are often used in molecular monitoring of hematological malignancies, and a quantification of residual disease is important for further prognosis. Here, the limiting dilution methodology and the multiplex IgH/ras PCR are proposed as approaches to molecular monitoring of NHLs. Applying the limiting dilution methodology as a simple dose-response assay for the translocation t(14,18) and CDR3 clonal rearrangement of IgH, critical amounts of total cells determined with stored consecutive diagnostic samples in the same PCR run are compared. Assuming that specific targets are diluted proportionally in dilution of total genomic DNA, the samples showing lower critical concentrations of total DNA are considered as containing higher portion of cells possessing the specific disease marker and vice versa. So far, the correlation of results with the disease outcome confirmed that this simple semi-quantitative approach may in some cases substitute laborious precisely quantifying techniques in the monitoring of the disease. In optimized multiplex IgH/ras PCR co-amplifying clonal CDR3 rearrangement of IgH and the codon 61 of Hras 1 gene, the amount of CDR3 product as the disease marker is related to the ras product as a standard marker of all cells, and quantitative results are obtained by software analyses of detecting gels. Presumably, both approaches may provide clinically useful information on the disease activity and treatment outcome.