Context.—Previous studies have shown that loss of the X chromosome is involved in the carcinogenesis of certain human malignancies.

Objective.—To determine whether X-linked allelic losses occur during bladder tumorigenesis and whether such losses involve the active or the inactive X chromosome.

Design.—We analyzed the deletion status of the X-linked human androgen receptor gene locus in 6 female patients who underwent radical cystectomies for muscle-invasive urothelial carcinoma of the urinary bladder. Four patients had coexisting urothelial carcinoma in situ. Analysis for inactivation of the X chromosome was carried out in parallel.

Results.—Three cases were informative. Invasive tumor samples showed loss of heterozygosity involving the active allele at the androgen receptor locus in all 3 positive cases, whereas carcinoma in situ showed nonrandom X chromosome inactivation but not allelic deletion.

Conclusions.—Our data suggest that allelic loss of the activated X chromosome is involved in bladder carcinogenesis and cancer progression.

Bladder cancers provide an important model for study of genetic alterations during tumor progression and metastasis.1–3 It is speculated that multiple genetic changes in oncogenes and tumor suppressor genes are necessary for transformed cells to acquire a malignant phenotype. Loss of heterozygosity (LOH) studies suggest that deletions of chromosome 9p and 9q and the p53 gene occur frequently during the development and progression of urothelial carcinoma of the urinary bladder.1,2,4–8 Allelic losses involving the X chromosome have been associated with certain human malignancies.9–14 Cheng et al9 conducted a DNA methylation status analysis that focused on the androgen receptor (AR) locus and found that the inactive X chromosome was lost in ovarian tumors of low malignant potential along with LOH at the AR locus. Whether allelic losses involving the X chromosome are associated with bladder carcinogenesis is unknown. In this study, we sought to determine whether X-linked allelic losses occur during bladder tumorigenesis and whether such losses involve the active or the inactive X chromosome.

We studied 6 female patients who underwent cystectomies for muscle-invasive urothelial carcinoma of the bladder. Four patients had coexisting urothelial carcinoma in situ. Samples of carcinoma from different areas of the same tumor were obtained from 5-μm histologic sections prepared from formalin-fixed, paraffin-embedded blocks. Approximately 400 to 600 cells were microdissected under direct light microscopic visualization (BH2; Olympus, Tokyo, Japan) as previously described (Figure 1).15,16 Separately microdissected nonneoplastic tissue from the same section was used for the control samples. Samples were evaluated for LOH at the AR locus located at Xq11-12. X chromosome inactivation was performed in parallel as described previously.17,18 Briefly, the dissected cells were placed in a 15-μL aqueous solution consisting of 10 mM Tris, 1 mM EDTA, 1% Tween 20, and 0.2 mg/mL proteinase K (pH 8.3) and incubated overnight at 37°C. The solution was boiled for 10 minutes to inactivate the proteinase K and used directly for subsequent clonal analysis without further purification. Eight-microliter aliquots of the DNA extract were digested overnight at 37°C with 1 U of HhaI restriction endonuclease (New England Biolabs Inc, Beverly, Mass) in a total volume of 10 μL. Equivalent aliquots of the DNA extracts were also incubated in the digestion buffer without the HhaI endonuclease as control reactions for each sample. After the incubation, 3 μL of digested or nondigested DNA was amplified in a 25-μL polymerase chain reaction (PCR) cocktail that contained 0.1 μL of 32[P]α-labeled deoxyadenosine triphosphate (3000 Ci/mmol), 4 μM AR-sense primer (5′-TCCAGAATCTGTTCCAGAGCGTGC-3′), 4 μM AR-antisense primer (5′-GCTGTGAAGGTTGCTGTTCCTCAT-3′), 4% dimethylsulfoxide, 2.5 mM MgCl2, 300 μM deoxycytidine triphosphate, 300 μM deoxythymidine triphosphate, 300 μM deoxyguanosine triphosphate, 300 μM deoxyadenosine triphosphate, and 0.13 U Taq DNA polymerase (Perkins-Elmer Corporation, Norwalk, Conn). Each PCR amplification had an initial denaturation step of 95°C for 8 minutes that was followed by 32 cycles at 95°C for 40 seconds, 63°C for 40 seconds, and 72°C for 60 seconds and, finally, a single final extension step at 72°C for 10 minutes. The PCR products were then diluted with 4 μL of loading buffer containing 95% formamide, 20 mM EDTA, 0.05% bromphenol blue, and 0.05% xylene cyanol FF (Sigma Chemical Company, St Louis, Mo). The samples were heated to 95°C for 5 minutes and then placed on ice. Three microliters of the reaction mixture was loaded onto 6.5% polyacrylamide denaturing gels without formamide, and the PCR products were separated by electrophoresis at 1600 V for 4 to 7 hours. The bands were visualized after autoradiography with Kodak X-OMAT AR film for 8 to 16 hours.19–22 

The cases were considered informative if 2 AR allelic bands were detected after PCR application in normal control samples. Taking advantage of a CAG-nucleotide repeat and several methylation-sensitive HhaI endonuclease sites on exon 1 of the AR gene (AR locus), the active X chromosome is cleaved after methylation-sensitive HhaI digestion and therefore cannot be amplified by the PCR primers we used. The inactive AR allele is preserved after the HhaI digestion and is therefore amplified by PCR. Polyclonal populations of cells are composed of a mosaic of cells with equal numbers of maternal-derived or paternal-derived active X chromosomes. In tumor samples, nonrandom X chromosome inactivation was defined as the complete or nearly complete absence of an AR allele after HhaI digestion, indicative of a monoclonal population.17,18 LOH was defined as the presence of a single allelic band without HhaI digestion.

We analyzed the deletion status of the X-linked human AR gene locus in 6 female patients. Three cases were informative (Table). Muscle-invasive tumors showed LOH of the AR locus at Xq11-12 in these 3 positive cases. In 2 of these 3 cases, carcinoma in situ showed nonrandom inactivation of the X chromosome (case 2, Figure 2, A; case 3, Figure 2, B) but not allelic loss. In cases 1 and 2, invasive tumor samples from different areas of the same tumor showed different patterns of allelic loss, whereas in case 3, they showed the same pattern of allelic loss at the AR locus (Figure 2). The results of our study are summarized in the Table.

Clonal evolution of transformed cell populations requires one or more genetic changes to obtain growth advantages over adjacent normal cells and to form a clinically detectable tumor.23 This study provides evidence that allelic loss in the active X chromosome is involved in bladder tumorigenesis and cancer progression in some cases. Allelic loss at the AR locus involved the active allele in each tumor sample analyzed, whereas carcinoma in situ showed nonrandom inactivation of the X chromosome instead of allelic loss. It has been postulated that muscle-invasive urothelial carcinoma develops from carcinoma in situ.2,3,24 

Experimental data appear to support the notion that a deletion of the active X-linked allele participates in tumorigenesis. Wang et al25 reported that microcell fusion of the normal Chinese hamster embryo (CHE) X chromosome into an immortal male CHE cell line (Ni-2/TGR) with an X deletion (Xq1) yielded senescence of all X recipient clones, whereas the normal human X chromosome induced senescence in 19% of these microcell hybrids. These data suggest that both human and CHE cells possess similar X-linked genetic activities that regulate the process of cellular senescence, important in the control of cell proliferation. Klein et al26 found that senescence in a similar cell line with an Xq deletion was reduced to 50% when Chinese hamster X chromosomes were transferred from later-passage A9 cells. Full senescing activity of the intact hamster X chromosome was restored by treatment of the donor mouse cells with 5-azacytidine, which induced demethylation of DNA, indicating gene silencing of X-linked alleles by DNA methylation. In previous LOH studies, LOH at the AR locus was also reported in several human solid tumors.9,10 Taken together, these data suggest the existence of a candidate gene in the chromosome Xq11-12 region whose function is important in cell growth control.

X chromosome inactivation is a well-characterized developmental event accompanied by the dense methylation of large numbers of CpG island genes,27,28 and this is known to contribute to the effectiveness of gene silencing.29 DNA hypermethylation of tumor suppressor genes is one of the primary inactivating events contributing to tumorigenesis and cancer progression.30–34 On the basis of Knudson's “2-hit” model of tumor formation,23 Myohanen et al35 and Esteller et al introduced a modification in which one allele of a tumor suppressor gene is a wild type but is silenced by hypermethylation, whereas the second allele is either mutated or lost. This new version of the “2-hit” genetic model for cancer progression may have important biologic and therapeutic implications. Reactivation of dormant genes using inhibitors of DNA methylation or demethylation agents may restore cell growth control.30,31,36 

Loss of the AR has been reported in urothelial carcinoma of the urinary bladder.37,38 Nevertheless, the AR itself may not be the target of the LOH on Xq11-12. Androgen has been shown to promote bladder tumorigenesis. Males are several times more at risk than females.39 In animal models, androgen plays an important role in bladder tumorigenesis induced by carcinogens.40–42 In this case, the loss of AR would suppress tumorigenesis. One explanation for the loss of the active allele at the AR locus is that, instead of the AR itself, LOH at Xq11-12 involves other tumor suppressor gene(s) closely linked to the AR.9 

It is evident that tumorigenesis is a multistep, dynamic process involving multiple gene loci. Our data suggest that the allelic loss of the activated X chromosome is implicated in bladder cancer progression.

This research was approved by the Indiana University Institutional Review Board.

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Author notes

Reprints: Liang Cheng, MD, Department of Pathology and Laboratory Medicine, Indiana University Medical Center, University Hospital 3465, 550 N University Blvd, Indianapolis, IN 46202 ([email protected])