Comparison of the BLCA-4 Gene Sequence Between Human Bladder Carcinoma and N-Methyl-N-Nitrosourea Induced Rat Bladder Carcinoma

Comparison of the BLCA-4 Gene Sequence Between Human Bladder Carcinoma and N-Methyl-N-Nitrosourea Induced Rat Bladder Carcinoma
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Title:Comparison of the BLCA-4 Gene Sequence Between Human Bladder Carcinoma and N-Methyl-N-Nitrosourea Induced Rat Bladder Carcinoma
Author/Abstract:
John Kenneth B. Domingo, MD; Josefino R. Castillo, MD, MSc; Jason L. Letran, MD and David T. Bolong, MD
 
Section of Urology, Department of Surgery, University of Santo Tomas Hospital

Objectives: Possible sources of drugs need to undergo stringent testing before any clinical trials are undertaken. Vital to this is the testing of these drugs on animals. The need for a homogenous animal xenotype to humans is not only necessary to understand biologic genetic factors that influence the phenotypic characteristics of the disease process but also serves as a basis for developing rational interventional strategies. This research aims to explore the BLCA-4 gene sequence of MNU induced urinary bladder cancer in rats and urinary bladder cancer in humans.i

Materials and Methods: Transitional cell carcinoma was induced in 3 male, age matched Wistar rats through intravesical instillation of 0.2 ml of N-methyl-N-nitrosourea and were sacrificed after 21 days. The urinary bladders of the rats were collected. Portions were sent for histopathologic studies by a veterinarian pathologist and the mucosa was scraped from the remaining tissues for further testing. Samples of 1.0 cm x 1.0 cm of human bladder tissue were also collected during the process of transurethral resection of bladder tumor with its mucosa scraped off for further testing. The genomic DNA from the collected urinary bladder mucosa were isolated using the Wizard® Genomic DNA Purification Kit. The isolated DNA were subjected to polymerase chain reaction with the use of degenerate primers consisting of BLCA-4 forward primer 5'-GAAATATCTAACTCAACGCCGGC-3' and BLCA-4 reverse primer 5'-GTCTACGAAGACATAATGCAAAAG-3'. The amplicons generated were subjected to gel electrophoresis. The samples were sent for purification and sequencing with the results run under the Bioedit® version 5.0.6 software and then aligned using Basic logical alignment search tools for nucleotides (BLASTN). Further verification was done using Nucleic Acid Sequence Maximum Likelihood Method (DNAmL) version 3.6a2.1

Results: All 3 rats developed transitional cell carcinoma, one human bladder cancer was labelled urinary bladder transitional cell carcinoma while the other was urinary bladder adenocarcinoma. There was successful isolation of genetic material from all specimen indicated by the production of bands. Five out of the 14 pair-wise alignment of the unedited sequences showed favorable results while only 3 out of the 14 pair-wise alignment of the edited sequences showed favorable results. Global alignment using the Bioedit® software showed similarity of all pair-wise alignment. Final verification study applied was Nucleic Acid Sequence Maximum Likelihood Method (DNAmL) version 3.6a2.1 which showed that the subjects are highly related.

Conclusion: The BLCA-4 marker gene expressed by humans and rats with urinary bladder cancer showed similar gene sequence. Thus, N-methyl-N-nitrosourea induced urinary bladder cancer in male wistar rats are good models for research testing.

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