Detecting homozygous deletions in the CDKN2A(p16(INK4a))/ARF(p14(ARF)) gene in urinary bladder cancer using real-time quantitative PCR.
OBJECTIVE
9p21 is a major target in the pathogenesis of human urinary bladder cancer. The locus harbors the CDKN2A/ARF tumor suppressor gene, which encodes two cell cycle regulatory proteins cyclin dependent kinase 2A (p16(INK4a)) and alternate reading frame (p14(ARF)). We have designed a real-time quantitative PCR (QPCR) application to study homozygous deletion (HD) of CDKN2A/ARF in 186 urinary bladder cancer patients.


METHODS
Real-time QPCR, based on simultaneous amplification of ARF and a reference gene, GAPDH, was developed and evaluated in three melanoma cell lines with HDs at the CDKN2A/ARF locus (IGR-1, SK-MEL-5, and WM-266-4). In addition, loss of heterozygosity was analyzed at the D9S942, D9S1748, and D12S99 markers. Mutation analysis of the CDKN2A/ARF gene was performed using single-strand conformational polymorphism and sequencing. Results from the present investigation were combined with previous p53 analysis of the same urinary bladder neoplasms.


RESULTS
Real-time QPCR analysis showed 26 (14%) HDs, 22 (12%) hemizygous deletions, and 3 (2%) multiple duplications. Loss of heterozygosity was determined in 30 (22%) cases at the D9S942 locus, which is located between E1alpha and E1beta of the CDKN2A/ARF gene. No association was established between occurrence of genetic aberrations at 9p21 and tumor stage or grade, supporting previous suggestions that CDKN2A/ARF inactivation is an early event in bladder carcinogenesis.


CONCLUSIONS
We have established a fast and efficient method for detection of HDs. Our data support the notion that inactivation, including HDs, of CDKN2A/ARF is an early event in transitional cell carcinoma. We observed separate and specific targeting of the CDKN2A and ARF genes, respectively, and that simultaneous inactivation of ARF and p53 occurs.
Berggren
Petra
P
Department of Biosciences at Novum, Karolinska Institute, 141 57 Huddinge, Germany.
Kumar
Rajiv
R
Sakano
Shigeru
S
Hemminki
Liina
L
Wada
Takashi
T
Steineck
Gunnar
G
Adolfsson
Jan
J
Larsson
Per
P
Norming
Ulf
U
Wijkström
Hans
H
Hemminki
Kari
K
D002295:Carcinoma, Transitional Cell / Q000235:genetics; D005091:Exons; D017353:Gene Deletion; D019942:Genes, p16; D016158:Genes, p53; D005819:Genetic Markers; D006720:Homozygote; D006801:Humans; D018895:Microsatellite Repeats; D008957:Models, Genetic; D009154:Mutation; D016366:Open Reading Frames; D011110:Polymorphism, Genetic; D018807:Polymorphism, Single-Stranded Conformational; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D001749:Urinary Bladder Neoplasms / Q000235:genetics*
D005819:Genetic Markers; D012333:RNA, Messenger