A hMLH1 genomic mutation and associated novel mRNA defects in a hereditary non-polyposis colorectal cancer family.

Hereditary non-polyposis colorectal cancer (HNPCC), or Lynch syndrome I, is responsible for as high as 10% of all colorectal cancers (CRCs) newly diagnosed in any given year. This disorder has an autosomal dominant inheritance pattern and is almost fully penetrant (>85%). It occurs when there is a mutation in any one of six mismatch repair genes: hMLH1, hMSH2, hPMS1, hPMS2, hMSH3 and hMSH6. Mutations in these genes allow mistakes in tumor suppressor genes and oncogenes to accumulate which eventually leads to cancer. The founder of an HNPCC family in the Creighton University Hereditary Cancer Institute database was known to produce truncated hMLH1 protein, a product of one of the aforementioned mismatch repair genes. Lymphoblasts were isolated from ten members of this HNPCC family (six affected and four unaffected) and two persons from outside this family (both unaffected controls). RNA and DNA were purified from these lymphoblasts which had been transformed by the Epstein-Barr virus (EBV). The hypothesis was that a mutation in the hMLH1 gene perpetuated defects in its mRNA and functional protein. hMLH1 RNA transcripts were detected in reverse transcriptase polymerase chain reactions (RT-PCR) whereby total poly A(+) RNA was converted to a complementary DNA (cDNA), amplified using hMLH1 specific primers, purified and cycle sequenced. Likewise, DNA was employed as template for PCR amplification of hMLH1 exons; PCR products were then directly cycle sequenced. Affected family members were found to produce hMLH1 mRNA lacking exons 6 and 7 (and wild-type mRNA). A splicing mutation at 546--2 (two bases 5' to exon 7) was located in the genomic DNA samples from the six family members with the HNPCC phenotype. This mutation caused deletion of exon 7 from the mRNA. None of the four unaffected family members or the two unaffected persons outside of this family had the above defects in their hMLH1 mRNA and DNA.

The inhibition of bovine carbonic anhydrase by saccharin and 2- and 4-carbobenzoxybenzene sulfonamide.

The present work demonstrates that the high-activity zinc metalloenzyme, carbonic anhydrase (CA II) from bovine erythrocytes is inhibited by the cyclic sulfimide, saccharin, and 2- and 4-carbobenzoxybenzene sulfonamide. A spectrophotometric method was employed to monitor the enzymatically catalyzed hydrolysis of p-nitrophenyl acetate by following the increase in absorbance at 410 nm which accompanies p-nitrophenoxide/p-nitrophenol formation. The more rapid enzymatic hydration of CO2 was monitored by using a stopped-flow spectrophotometer as well as by a modified colorimetric method of Wilbur and Anderson. The studies show that, at a given molar ratio of inhibitor to enzyme, the degree of inhibition of the enzymaic hydration of CO2 and hydrolysis of p-nitrophenyl acetate by the inhibitory compounds is essentially the same. Kinetic analyses were made at 25.0 degrees at pH 6.5 (MES buffers), pH 6.9 (HEPES buffers) and pH 7.9 (HEPES buffers) with ionic strength regulated by the addition of appropriate quantities of sodium sulfate. Lineweaver-Burk plots were used to evaluate apparent inhibition constants for each of the three inhibitors. For all the inhibitors studied, inhibition appears to be mixed (competitive/noncompetitive). For saccharin in the presence of sodium sulfate, the extent of inhibition is considerably decreased. It was found for the three inhibitors that the inhibitory potency decreases with increasing pH, and that the inhibitory potency is extremely sensitive to the shape of these rather closely related molecules. For example, apparent inhibition constants for the enzymatic hydrolysis of p-nitrophenyl acetate at pH 6.9 were Ki (saccharin) = 0.20 mM, Ki (2-carbobenzoxybenzene sulfonamide) = 0.54 mM and Ki (4-carbobenzoxybenzene sulfonamide) = 1.6 microM. For the enzymatic hydration of CO2 at pH 6.9, 0.10 mM saccharin caused 50% inhibition while 7.0 nM 4-carbobenzoxybenzene sulfonamide resulted in 50% inhibition. The results suggest that sulfonamide inhibition is caused by formation of a monodentate ligand at the zinc ion of the enzyme active site and that the more linear 4-carbobenzoxybenzene sulfonamide is better able to enter a conical enzyme active site than is 2-carbobenzoxybenzene sulfonamide or saccharin.