The use of dynamic size-sieving capillary electrophoresis and mismatch repair enzymes for mutant DNA analysis.

The detection of base pair mismatches in limiting amounts of DNA is important in the early diagnosis of cancer and other genetic diseases. The specific type and exact location of a bp mismatch in certain genes can yield information on the likelihood of a patient developing a genetic disease, as well as the severity of the disease. We demonstrate two methods of specific DNA point mutation detection and identification that involve the integration of mismatch repair cleavage enzyme analyses with dynamic size-sieving capillary electrophoresis. The mismatch repair cleavage enzymes employ the very mechanism that a cell uses in its own mismatch recognition and repair systems. One analysis employs an isothermal signal amplification process, and the other involves polymerase chain reaction (PCR) (Hoffmann-LaRoche, Nutley, NJ, U.S.A.) for amplification of the DNA. Separation of the DNA fragments using dynamic size-sieving CE yields a cleaved fragment, providing definitive evidence of a bp mismatch. The specificity and sensitivity of the assay are facilitated by the detection of fluorescently labeled DNA fragments using laser-induced fluorescence detection; picogram quantities of a target DNA can be analyzed reproducibly.

RecA-assisted rapid enrichment of specific clones from model DNA libraries.

An approach to library screening is being developed, in which the desired clone is "fished" out of a mixture of all the recombinants in a library with a RecA-coated probe. In the current embodiment of this method, we used as a probe the (+) strand of an M13 phage containing a fragment of the human albumin gene and a (dA)49 stretch. We screened a library of two plasmids, one containing the same albumin fragment as the probe, and one heterologous to the probe in 50-100 fold molar excess. The plasmids were linearized. Probe and library were reacted in the presence of RecA, the mixture was loaded onto an oligo(dT) column, which retained the probe-target complex by base-pairing to the dAs of the probe, the uncaptured plasmids were washed, and the probe-target complex was released from the column, religated and propagated into E. coli. Recovery of the homologous target was 15-28%, and enrichment for the homologous plasmid was 200 to 400-fold. This approach may provide a general method for expedited DNA library screening.

Prostaglandin E2 down-regulates the expression of HLA-DR antigen in human colon adenocarcinoma cell lines.

Prostaglandins (PG) have been implicated in the pathogenesis of cancer and play an important role in immune regulation. Colon cancer is associated with elevated levels of PGE2, while aspirin, the prototypical inhibitor of PG synthesis, appears to reduce the incidence of colon cancer by 50%. We have observed that in human colon cancer the expression of HLA class I and II antigens is reduced or lost; loss of HLA antigens is suspected to be a mechanism by which the malignant cell escapes the immune surveillance. We investigated the effect of these eicosanoids on the expression of HLA antigens in human colon adenocarcinoma cell lines. PGE2 down-regulated the expression of the class II antigen HLA-DR in SW1116 cells (65% reduction at 2.8 x 10(-8) M). This effect was dose- and time-dependent, reversible, and specific (PGF2 alpha and LTB4 had no effect; the expression of carcinoembryonic antigen and class I genes were not affected). Aspirin induced the expression of HLA-DR in HT29 cells, a cell line not expressing constitutively HLA-DR. The reduction of HLA-DR by PGE2 was accompanied by reduced messenger RNA (mRNA) levels of HLA-DR alpha and reduced transcription of the corresponding gene. In contrast to HLA-DR, none of these three eicosanoids affected the expression of HLA class I genes, as assessed via determination of protein expression by fluorescence flow cytometric analysis and evaluation of the corresponding class I mRNA levels. We conclude that PGE2 specifically down-regulates the expression of HLA-DR, while it does not affect the expression of class I antigens.(ABSTRACT TRUNCATED AT 250 WORDS)

Lithocholic acid inhibits the expression of HLA class I genes in colon adenocarcinoma cells. Differential effect on HLA-A, -B and -C loci.

Loss of HLA antigen expression is considered to be one of the mechanisms whereby tumor cells escape immune surveillance. We recently observed reduced or lost expression of HLA antigens during human colon carcinogenesis. We studied the effect of bile acids (BAs), long implicated in the pathogenesis of colon cancer, on the expression of HLA class I antigens in human colon adenocarcinoma cells. Lithocholic acid (LCA) decreased by 42% the expression of HLA class I antigens on the surface of these cells. This dose-dependent reduction was specific for both the target genes and the chemical structure of LCA, and was not evident in cultured liver cells. None of the other BAs that were tested manifested this effect. LCA, and to a lesser extent deoxycholic acid (DCA), decreased steady-state HLA class I mRNA levels. LCA decreased the rate of transcription of HLA-B (64%) and HLA-C (87%) but not HLA-A; DCA had a similar but less pronounced effect. In transient gene expression (CAT assays) experiments, we evaluated the role of a 0.6-0.7 kb EcoRI/XbaI sequence from the 5' flanking region of HLA-A2, -B7 and -Cw7 genes in the regulation of class I gene expression by LCA. LCA down-regulated by 70% the expression of the reporter gene for all three genes. We interpret these results as indicating a differential regulation of the three HLA loci by LCA. Our findings, demonstrating a profound effect of LCA on HLA class I gene regulation, raise the possibility that such a mechanism may be operative in vivo.