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Journal of Cancer Research and Clinical Oncology logoLink to Journal of Cancer Research and Clinical Oncology
. 2004 Dec 14;131(3):157–162. doi: 10.1007/s00432-004-0638-8

Association between clinical characteristics and expression abundance of RTKN gene in human bladder carcinoma tissues from Chinese patients

Jie Fan 1,✉, Li-Jie Ma 2, Shu-Jie Xia 1, Long Yu 2, Qiang Fu 2, Chao-Qun Wu 2, Xing-Hua Huang 2, Jian-Ming Jiang 2, Xiao-Da Tang 1
PMCID: PMC12161221  PMID: 15599595

Abstract

Purpose

Bladder carcinoma is the most common urological malignancy in China. Gene mutation may be one of causes of carcinogenesis in the cancer. We therefore investigated the mRNA expression of RTKN gene in clinic malignant bladder carcinoma and explored the relationship between the novel gene and the cancer.

Methods

Total RNA was extracted from 33 surgically resected specimens of bladder carcinoma and 19 specimens of tumor-free bladder tissues. After the optimal reverse-transcription polymerase chain reaction condition was established, the mRNA expression levels of the RTKN gene in the lesions and tumor-free bladder tissues were examined semiquantitatively, and the relationships between expression levels of RTKN and clinical pathological features were analyzed.

Results

The expression of RTKN gene mRNA in 33 human bladder carcinoma tissues was significantly higher than that in 19 human tumor-free bladder tissues (0.937±0.103 vs. 0.350±0.082). The average ratio of RTKN expression in neoplasms to that in tumor-free bladder tissues was 0.350±0.164. Based on this ratio the 33 patients were divided into three groups: a downregulated expression group (n=2), an upregulated expression group (n=22), and an unchanged group (n=9). Although the χ2 test demonstrated a statistically nonsignificant differences in RTKN expression between tumor stages Ta, T1, and T2 overall in the 33 human bladder carcinoma, the t test showed that there were statistically significant differences between solitary and multiple tumors, between the paired group aged younger or older than 70 years in 27 de novo bladder carcinoma patients, and between the groups with tumor larger or smaller than 2.25 cm3.

Conclusions

These results suggest that the RTKN gene is involved in bladder carcinogenesis and progression in bladder carcinoma, indicating that RTKN gene could be a molecular target in cancer therapy.

Keywords: RTKN gene, Bladder neoplasm, Carciogenesis, Reverse transcription polymerase chain reaction

Introduction

Urinary bladder carcinoma is the most common malignancy of urological tumors in China, and its frequency has been rapidly increasing especially in recent years. Investigating the carcinogenesis of the cancer is important not only for prevention and prognosis but also for treatment. It has been hypothesized that oncogene changes are the cause of the cancer. Ras was the first human oncogene cloned in human bladder carcinoma tissue and found to be involved in carcinogenesis of bladder carcinoma (Przybojewska et al. 2000), and Rho is a member of the Ras superfamily of small GTP-binding protein (Van Aelst and D’Souza-Schorey 1997). The Rho proteins are a class of small molecular GTPases that regulate multiple fundamental cellular processes by mediating the G protein coupled receptor signaling pathway. The Rho gene has been studied in many tumors. Data on the biological relevance of the rho genes in pancreatic carcinogenesis suggest that elevated expression of the rhoC gene is involved in the progression of pancreatic carcinoma independent of K-ras gene activation (Suwa et al. 1998). In ovarian carcinoma some reports show that upregulation of Rho GTPases is important in the tumor progression of ovarian carcinoma, and that Rho family proteins could be a molecular target in cancer therapy (Horiuchi et al. 2003). At the protein and mRNA levels in breast tumors results show that the levels of RhoA, RhoB, Rac1, and Cdc42 proteins are largely enhanced in all tumor samples analyzed (n=15) compared to normal tissues. Overall the data show that (a) Rho proteins are overexpressed in breast tumors, (b) overexpression is not regulated at the mRNA level, (c) the expression level of RhoA-like proteins is correlated with malignancy, and (d) Rho proteins are not altered by mutation in breast tumors (Fritz et al. 2002). However, immunohistochemistry shows that RhoC is specifically expressed in invasive breast carcinomas capable of metastasizing, and it may be clinically useful in patients with tumors smaller than 1 cm to guide treatment (Kleer et al. 2002).

The relationship between neoplasms of the genitourinary tract and the rho gene was reported only in recent years. One finding suggests that RhoA is involved in testicular germinal epithelial carcinogenesis and progression in testicular germ cell tumor, indicating that RhoA may be a useful prognostic marker for progression in testicular germ cell tumor (Kamai et al. 2001). At levels of mRNAs of RhoA and Rho kinase in tumor tissues these findings (Kamai et al. 2002) suggest that the RhoA/Rho kinase pathway is involved in the progression of testicular germ cell tumor. This pathway might be a molecular target for new treatment strategies for this disease. Kamai et al. (2003) also studied the association of the Rho/ROCK pathway with invasion and metastasis of bladder cancer; their conclusion was that the Rho/ROCK pathway apparently involved in occurrence and progression of bladder cancer may be valuable prognostic markers. At our laboratory we first reported a novel human cDNA containing an intact open reading frame that encodes 544 amino acids. This was regarded as a human homologue of the mouse Rhotekin and termed RTKN (Fu et al. 2000). To determine whether the novel human gene is involved in the carcinogenesis of bladder carcinoma we used semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) to detect changes in RTKN gene mRNA expression in human bladder carcinoma and tumor-free urinary bladder tissues.

Materials and methods

Tumor samples

Fresh surgical specimens of bladder cancer tissues, diagnosed histologically as bladder carcinoma from 33 Chinese patients were obtained from the Department of Urology, Shanghai First People’s Hospital, affiliated with Shanghai Jiaotong University, China (Table 1). Specimens of control normal bladder tissues were obtained from pathologically tumor-free urinary bladder tissues, including three bladder tissues from cadavers, five when performing suprapubic prostatectomy, two from radical cystectomy of bladder carcinoma patients, and nine from partial cystectomy of bladder carcinoma patients. The materials were immediately frozen in liquid nitrogen and stored at −80°C.

Table 1.

Clinical characteristics of 33 bladder carcinoma patients

Case no. Age (years) Sex Pathological type of bladder carcinoma Grade AJCC stage Size (cm3) Number Recurrence
1 40 M Transitional cell carcinoma II T2 42.00 Multiple –
2 70 F Transitional cell carcinoma I T1 0.25 Solitary –
3 83 M Transitional cell carcinoma II T1 6.00 Multiple –
4 49 M Transitional cell carcinoma III T1 3.00 Solitary –
5 71 M Transitional cell carcinoma I T2 7.50 Multiple Recurrence
6 56 M Adenocarcinoma I T1 0.80 Multiple –
7 58 M Transitional cell carcinoma II T2 14.00 Multiple –
8 67 M Transitional cell carcinoma I T1 2.25 Multiple –
9 87 F Transitional cell carcinoma I T1 0.25 Multiple –
10 73 M Transitional cell carcinoma I T1 3.00 Multiple –
11 83 M Transitional cell carcinoma III T2 15.00 Solitary –
12 48 M Transitional cell carcinoma II T2 9.00 Multiple Recurrence
13 57 F Transitional cell carcinoma III T2 5.00 Solitary –
14 74 M Transitional cell carcinoma I Ta 1.00 Multiple Recurrence
15 69 M Transitional cell carcinoma I T1 2.25 Multiple –
16 50 M Adenocarcinoma II T2 9.00 Solitary –
17 72 M Transitional cell carcinoma II T1 1.00 Multiple Recurrence
18 72 M Transitional cell carcinoma III T2 2.40 Multiple Recuurence
19 57 M Transitional cell carcinoma II T1 2.25 Multiple Recurrence
20 77 M Transitional cell carcinoma I Ta 2.25 Solitary –
21 55 M Transitional cell carcinoma II T1 2.25 Multiple –
22 76 M Transitional cell carcinoma II T1 4.00 Multiple –
23 82 M Transitional cell carcinoma II T1 60.00 Multiple –
24 75 M Transitional cell carcinoma II T1 10.50 Multiple –
25 79 F Transitional cell carcinoma III T1 3.75 Solitary –
26 68 M Transitional cell carcinoma I Ta 0.50 Multiple –
27 56 M Transitional cell carcinoma II T1 1.50 Multiple –
28 89 M Transitional cell carcinoma II T1 9.00 Solitary –
29 80 F Transitional cell carcinoma II T2 6.00 Multiple –
30 70 M Transitional cell carcinoma II T1 2.25 Solitary –
31 42 F Transitional cell carcinoma I Ta 1.50 Solitary –
32 50 F Transitional cell carcinoma III T2 12.00 Solitary –
33 65 M Transitional cell carcinoma II T2 0.25 Solitary –
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Total RNA isolation

Total RNA was extracted from bladder carcinoma and normal tissues using Trizol reagent (Life Technologies) according to the manufacturer’s protocol.

Primers for RT-PCR amplification

The sequences of oligonucleotide primers for RT-PCR for the RTKN gene at expected sizes of their RT-PCR products are as follows:

  • RTKN C: 5′-AAA GGT GCT GGC ATA GGA TCT GC-3′

  • RTKN D: 5′-TGG TTG ATG TGG GAG TCA CAA GG-3′, 334 bp

  • β2-MG C1 (Gussow et al. 1987): 5′-ATG AGT ATG CCT GCC GTG TGA AC −3′(sense primer)

  • β2-MG C2: 5′-TGT GGA GCA ACC TGC TCA GAT AC-3′, 284 bp

Semiquantitative RT-PCR analysis

cDNA was synthesized using 2 µg total RNA, Superscript II reverse transcriptase (Gibco BRL) and oligo(dT)15 (Promega) according to the manufacturer’s protocols. First-strand cDNA was synthesized by RT and followed by PCR amplification on FS-918 DNA Amplifier (Shanghai Fusheng Institute of Biotechnology). The concentration of templates was regulated to be about 1 µl per 25 µl PCR reaction volume by amplifying (60°C, 30 s) for 30 cycles. To determine the best number of cycles PCR was performed at 20–37 cycles. The PCR products from each cycle were run on 2% agarose gel electrophoresis photographed and then quantified by gel imaging system (GDS8000, UVP). The growth curve of the PCR products was made according to the amount of PCR products of different cycles (Ozaki et al. 2000). The best cycle was determined between the increased logarithmic phase and plateau phase β2-MG. They were 30 cycles for β2-MG C1C2 (60°C, 30 s). Afterwards, the RTKN genes were amplified by using same amounts of the diluted templates with β2-MG C1C2 as the control for RTKN. The RT-PCR products were separated on 2% agarose gels, then photographed and quantified.

Statistical analysis

The t-test was used to compare the levels of RTKN between bladder carcinoma and their tumor-free urinary bladder tissue. The t test and χ2 test were used to analyze the relationship between gene expression level and clinical characteristics.

Results

Analysis of expressional change in the RTKN gene in bladder carcinoma tissues

Semiquantitative RT-PCR was carried out to observe the expression of RTKN in 33 bladder carcinoma patients. First, according to the cumulative curve of the PCR products obtained with β2-MG C1C2 primer, the best numbers of cycles used in semiquantitative RT-PCR were determined between the increased logarithmic phase and plateau phase (Fig. 1A). The expression patterns of RTKN in other tissue samples were examined using the semiquantitative RT-PCR method and compared with the results from northern hybridization before at our laboratory. The results from the semiquantitative RT-PCR and northern hybridization were the same, which suggested that the RT-PCR method and conditions used in this study were reliable (Chen et al. 2003; Fu et al. 2000; Jiang et al. 2001). Therefore the expression of RTKN in 33 bladder carcinoma and 19 tumor-free bladder tissues was examined. The semiquantitative RT-PCR products of 33 bladder carcinoma samples were detected by gel imaging system (GDS8000, UVP) and statistically analyzed by t test. The level of β2-MG, as control, did not differ significantly (P=0.667) between bladder carcinoma tissues and the tumor-free tissues. The expression of RTKN in bladder carcinoma tissues was significantly upregulated (P=0.00062) compared with the tumor-free tissues. On the other hand, the ratio of RTKN to β2-MG was significantly increased (P=0.000272) in bladder carcinoma tissues against in the tumor-free tissues (Fig. 1C). We analyzed RTKN mRNA expression in 33 human bladder carcinoma and 19 human bladder tissues. Figure 1B shows the RT-PCR results of partial samples.

Fig. 1 A.

Fig. 1 A

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The best cycle of β2-MG and RTKN. The results suggest that both of the two gene’s best cycle is 30 cycles. B The expression of RTKN gene in bladder carcinoma tissues and tumor-free tissues. The figure shows that the RTKN gene is expressed in both of bladder carcinoma tissues and tumor-free tissues, but that in bladder carcinoma tissues it is expressed much more than in tumor-free tissues. C The best cycles of the RTKN gene’s PCR amplification. The level of β2-MG did not differ statistically significantly (P=0.667181) between bladder carcinoma tissues and the tumor-free tissues. The expression of RTKN in bladder carcinoma tissues was significantly upregulated (P=0.000620) compared with the tumor-free tissues. On the other hand, the ratio of RTKN to β2-MG was significantly increased (P=0.00027) in bladder carcinoma tissues against in the tumor-free tissues

Analysis of relationship between gene expression level and clinical characteristics

The results from individual analysis of the ratio of tumor to tumor-free tissues revealed that RTKN was upregulated in 22 of 33 (66.7%) bladder carcinoma tissues. As shown in Fig. 1B, RTKN was upregulated in 22 of the 33 cases and downregulated in 2 while no obvious change was observed for RTKN in 9 cases.

Based on the above findings the relationship between the clinical characteristic of these patients and RTKN expression level was analyzed further by stratifying these patients according to the clinical data pattern and comparing RTKN expression again by Student’s t test. The results show that the RTKN expression level of the multiple tumor (subgroup A1, n=15) is higher than that of the solitary tumor (A2, n=7) in patients with Ta or T1 stage (superficial disease), that the RTKN expression level of the tumor from patients aged under 70 years (subgroup B1, n=13) was greater in those aged 70 years or over (subgroup B2, n=14) in those primary patients, and that the RTKN expression level of tumors of size 2.25 cm3 or larger (subgroup C1, n=7) was greater than that in tumors of size less than 2.25 cm3 (subgroup C2, n=20) in those de novo patients (Fig. 2A). All differences in expression level in the above three paired subgroups are statistically significant (P<0.05). In addition to the three clinical characteristics, there were no significant associations between other clinical characteristics, such as sex, pathological type, pathological grade, stage, or recurrences, and the RTKN expression level of tumor.

Fig. 2 A.

Fig. 2 A

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t Test statistical analysis of association between clinical characteristics and expression abundance of RT-PCR amplification product density in 33 tissue samples of human bladder carcinoma patients. Among patients of superficial bladder carcinoma expression in the group with solitary tumors is greater than that in the group with multiple tumors. Among patients with de novo bladder carcinoma expression in the group aged 70 years or over is less than that in the group aged over 70, but expression in the group with tumor size of 2.25 cm3 or larger is greater than that in the group with tumor size smaller than 2.25 cm3. B Semiquantitative RT-PCR mRNA expression in 33 human bladder carcinoma patients

Discussion

As with Ras, Rho cycles between a GDP-bound inactive state and a GTP-bound active state. Rho proteins occupy key positions in many fundamental cellular processes (Reid et al. 1996). The conformational change in small GTPases has been studied in Ras by radiographic analysis of crystal structures of GDP-bound and GTP-bound forms of Ras. Rho also synergizes with and enhanced Ras-mediated DNA synthesis, Rho complemented Ras-mediated cell cycle progression by reducing the expression of P21waf/cip1 (Olson et al. 1998; Seasholtz et al. 1999). Recently Kamai et al. (2003) investigated the roles of Rho and ROCK (Rho’s best-characterized downstream effector Rho-associated serine-threonine proteinkinase) in bladder cancer and found that RhoA, RhoC, and ROCK were more abundant in tumors and metastatic lymph nodes than in nontumor bladder and uninvolved lymph nodes. High RhoA, RhoC, and ROCK expression were related to poor tumor differentiation muscle invasion and lymph node metastasis. Kaplan-Meier plots linked high RhoA, RhoC, and ROCK protein expression to shortened disease-free and overall survival (P<0.0001). By univariate analysis high RhoA, RhoC, and ROCK protein expression predicted shortened disease-free and overall survival. They concluded that the Rho/ROCK pathway apparently involved in occurrence and progression of bladder cancer may be valuable prognostic markers. However, other reported that elevated expression of the rhoC gene may be involved in the progression of pancreatic carcinoma independent of K-ras gene activation. We are therefore interested in the roles of small molecular GTPases in the carcinogenesis of bladder cancer.

The Rho proteins are a class of small molecular GTPases that regulate multiple fundamental cellular processes by mediating the G protein coupled receptor signaling pathway. Rhotekin, which is one of the downstream target molecules of Rho with a Rho binding motif class I domain, can inhibit endogenous or RhoGAP-stimulating Rho GTPase activity to regulate the signaling pathway. A novel human cDNA containing an intact open reading frame that encodes 544 amino acids has been identified (Fu et al. 2000); it was regarded as a human homologue of the mouse Rhotekin and termed RTKN. The RTKN gene was localized to chromosome 2p13 between markers D2S145 at 6.94 cR (LOD >12) and D2S286 at 8.12 cR (LOD >9.7) by radiation hybrid panel mapping. Compared with the bacterial artificial chromosome clone AC005041 sequence, there were 12 exons for the RTKN gene, and it spanned a 16.5-kb genomic region. The human Rhotekin (RTKN) cDNA is for the first time identified and reported (Fu et al. 2000). Therefore we reported the expression of RTKN gene in bladder carcinoma tissues for the first time. These findings show that the RTKN gene is significantly upregulated (P<0.001) in bladder carcinoma, which demonstrates that RTKN may be involved in the carcinogenesis of bladder carcinoma.

Our results show that the expression level of RTKN is significantly related with tumor size among de novo patients and tumor number among the superficial bladder tumor (Ta+T1 stage) patients (P<0.05). Some evidence implicates Rho in actin reorganization, cell motility, and cell growth, and transformation in non-smooth-muscle cell (Narumiya et al. 1997; Riddley and Hall 1994; Van Aelst and D’Souza-Schorey 1997), and Rho also functions as a signaling molecule in smooth muscle, in which it activates a mechanism that sensitizes the contractile machinery to Ca2+ (Sakurada et al. 2001). In genitourinary tract tumors some findings show that overexpression of Rho mRNA is associated with advanced stage I testicular germ cell tumor (Kamai et al. 2001), and RhoGDI2 mRNA expression was observed in a human bladder cancer cell line T24 and a more aggressive lineage related variant of it, T24T, suggesting that Rho activation plays a significant role in the metastatic cascade (Seraj et al. 2000). We therefore inferred that the RTKN protein may cause tumor progression. In the de novo bladder carcinoma patients aged under 70 years we observed that expression of the RTKN gene mRNA is higher than in those aged 70 years or over. Some other clinical data showed that younger bladder transitional cell carcinoma patients who ultimately underwent radical cystectomy had significantly lower disease-free survival, mainly due to a higher rate of distant metastases than in the older group (Yossepowitch and Dalbagni 2002). Therefore we inferred that bladder tumors with higher expression of RTKN gene in younger patients are more aggressive. Although there were no significant associations of other clinical characteristics, such as the pathological tumor type, grade, stage, or recurrences, with the expression level of RTKN, our RT-PCR results clearly show that RTKN is markedly upregulated, suggesting that RTKN is involved in the carcinogenesis of human bladder carcinoma.

In conclusion, the RTKN gene is a novel human gene, it is upregulation in human bladder carcinoma, suggesting that it is a novel human oncogene in human bladder carcinoma, and it may be another promising targeting protein for antineoplasmas (Fujiisawa et al. 1998; Reid et al. 1996). However, further investigation is required to test this hypothesis.

Footnotes

J. Fan and L.-J. Ma contributed equally to this manuscript

This work was supported by National Natural Science Foundation of China (no. 39900146)

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