Metastatic model for human prostate cancer using orthotopic implantation in nude mice.

BACKGROUND: Understanding the mechanism of prostate cancer metastasis is essential to the design of a more effective therapy. An effective therapy for this disease will depend on the development of a clinically relevant in vivo model. PURPOSE: We describe the development of such a model by using orthotopic implantation of human prostate cells in BALB/c nude mice. METHOD: We compared the tumorigenicity of and the incidence of metastasis of human prostate cancer PC-3M and LNCaP-FGC (LNCaP) cell lines subsequent to prostatic (orthotopic) or subcutaneous (ectopic) implantations in male nude mice. RESULTS: LNCaP cells produced tumors only in the prostate. Enhanced tumorigenicity at the orthotopic site was found for PC-3M cells. Lymph node metastases were observed in practically all mice given an injection of PC-3M cells in the prostate, but they were uncommon with subcutaneous injection of these cells. Bilateral orchiectomy did not alter the tumorigenicity of either PC-3M or LNCaP cells or the incidence of lymph node metastasis by PC-3M cells. LNCaP tumors in the mouse prostate (but not PC-3M tumors) elaborated detectable levels of human prostate-specific antigen (PSA) in the serum, even when tumors were small (1.5 mm in diameter). Immunohistochemistry analysis revealed the presence of the PSA marker in tissue sections of LNCaP but not of PC-3M tumors. CONCLUSIONS: The implantation of human prostate cancer cells in an ectopic environment does not permit expression of metastatic potential. In contrast, intraprostatic implantation does. IMPLICATIONS: These data suggest that the orthotopic injection of human prostate cancer cells into the nude mouse may provide a valuable model to study the biology and therapy of human prostate cancer.

Establishment of a human urachal adenocarcinoma cell line (KO-BT-1) and its chemosensitivity.

A new human urachal adenocarcinoma cell line (KO-BT-1) was established and characterized. It consisted of cuboidal, spindle, and polymorphic giant cells and continued to grow for more than 27 months without contact inhibition. Doubling time was about 15.5 h at the 70th passage. In nude mice the cells produced tumors, the histologies of which were similar to the original patient-derived tumor. Moreover, serum carcinoembryonic antigen level was elevated in the patient and the histological section of tumor formed in nude mice was stained with an antibody to carcinoembryonic antigen by peroxidase-antiperoxidase method. Electron microscopically, the cells covered with microvilli had cell-junction complexes. The chromosome number was aneuploid with a modal number of 60. At clinically achievable concentrations, doxorubicin, cis-platinum, mitomycin C, and 40487S which was an in vitro-active type of cyclophosphamide did not reduce colony formation to 30% or less of the control value; likewise in this patient chemotherapy had not been effective. In addition, among human recombinant tumor necrosis factor, alpha-interferon, and recombinant beta- and gamma-interferon, recombinant IFN-beta was most effective against this tumor. These results indicated that KO-BT-1 cells showed the similar chemosensitivity and properties to those of the original tumor, and might be useful in basic studies for the diagnosis, treatment, and etiology of urachal tumors.

Prognostic significance of circulating matrix metalloproteinase-2 to tissue inhibitor of metalloproteinases-2 ratio in recurrence of urothelial cancer after complete resection.

The relationship between the serum matrix metalloproteinase-2 (MMP-2):tissue inhibitor of metalloproteinases-2 (TIMP-2) ratio and disease recurrence was examined in 53 urothelial cancer patients with muscular invasion or with lymph node metastasis who underwent complete resection. The mean MMP-2:TIMP-2 ratio in 31 patients with recurrence was significantly higher than that in 22 patients without recurrence (P < 0.05). Disease-free survival of patients with high MMP-2:TIMP-2 ratios was extremely poor compared with that of patients with lower ratios (P < 0.01). Cox's multivariate analysis suggests that the serum MMP-2:TIMP-2 ratio would be a new independent prognostic indicator of urothelial cancer recurrence.

Enhanced inhibition of colony formation of human renal cell carcinoma in soft agar by the combination of alpha-difluoromethylornithine and recombinant gamma-interferon.

We studied the inhibitory activity of alpha-difluoromethylornithine (DFMO) and alpha-, recombinant beta-, and recombinant-gamma-interferons (alpha-, rec-beta-, and rec-gamma-IFNs) on in vitro growth of 3 established human urogenital tumors (KO-RCC-1 from renal cell carcinoma, Bewo from choriocarcinoma of the uterus, and HT-1197 from transitional cell carcinoma of the urinary bladder) and 16 primary renal cell carcinomas obtained by nephrectomy. Treatment with DFMO together with rec-IFN-gamma synergistically inhibited KO-RCC-1 cell growth in monolayer culture and in soft agar. The other two established cell lines were less susceptible to this treatment. Combination of DFMO and rec-IFN-gamma was more inhibitory than that of DFMO and either IFN-alpha or rec-IFN-beta. The polyamine content in KO-RCC-1 cells was decreased to a greater extent by combined treatment with DFMO and rec-IFN-gamma than that in Bewo and HT-1197 cells. The effect of these agents in 11 of the 16 primary renal cell carcinomas, which could show clonal growth in double layer soft agar, was examined. More than 50% inhibition of colony growth was seen in only one case (9%) treated with 5 mM DFMO alone and in 2 cases (18%) treated with rec-IFN-gamma alone (1,000 units/ml) but in 10 of the 11 cases (91%) with the combined treatment. Our results indicate that combined treatment with DFMO and rec-IFN-gamma can be more effective than that with either agent individually in inhibiting cell growth of human renal cell carcinoma in vitro.

Overexpression of Bcl-2 in bladder cancer cells inhibits apoptosis induced by cisplatin and adenoviral-mediated p53 gene transfer.

To investigate the effects of the expression of Bcl-2 protein in bladder cancer on the apoptosis induced by cisplatin or adenoviral-mediated p53 gene (Ad5CMV-p53) transfer, we transfected the bcl-2 gene into KoTCC-1, a human bladder cancer cell line that does not express the Bcl-2 protein. The Bcl-2-transfected KoTCC-1 (KoTCC-1/B) exhibited significantly higher resistance to both cisplatin and Ad5CMV-p53 transfer than did either the parental KoTCC-1 (KoTCC-1/P) or the vector-only transfected cell line (KoTCC-1/C). The flow cytometric analysis of the propidium iodide-stained nuclei and DNA fragmentation analysis after cisplatin or Ad5CMV-p53 treatment revealed DNA degradation in both KoTCC-1/P and KoTCC-1/C, whereas KoTCC1/B showed a marked inhibition of DNA degradation. Following the treatment with cisplatin or Ad5CMV-p53, the accumulation of p53 protein was highly detectable for a long period in KoTCC-1/B compared to that in KoTTC-1/P and KoTCC-1/C. Furthermore, the cisplatin and Ad5CMV-p53 treatments each reduced the volume of the subcutaneous tumors established in nude mice formed by KoTCC-1/P or KoTCC-1/C; in contrast, their reductive effects on the tumors formed by KoTCC-1/B were significantly suppressed. The intraperitoneal tumor cell implantation model revealed that the prognoses of mice injected with KoTCC-1/B were significantly inferior to those of the mice injected with either KoTCC-1/P or KoTCC-1/C after treatment with cisplatin or Ad5CMV-p53. These findings suggest that the expression of Bcl-2 in bladder cancer cells interferes with the therapeutic effects of cisplatin and Ad5CMV-p53 through the inhibition of the apoptotic pathway.

Molecular cloning and analysis of the 5'-flanking region of the human bone morphogenetic protein-6 (BMP-6).

We cloned a genomic fragment of the 5'-flanking region of the gene encoding bone morphogenetic protein-6 (BMP-6) and assessed its promoter activity. Primer extension revealed the presence of one major transcription start site 178 bp upstream of the translation start site. The promoter region lacked a canonical TATA box but did contain a GC-rich region. A putative tramtrack responsive element, a Drosophila transcriptional factor regulating the segment polarity, was found in the promoter region. Known steroid hormonal responsive elements, however, were not found. Although BMP-6 is classified as a member of the vgr-1 family, the structure of the promoter was similar to that of BMP-2 and 4.

Independent prognostic value of serum hepatocyte growth factor in bladder cancer.

PURPOSE: We retrospectively investigated whether the level of serum hepatocyte growth factor could predict the prognosis and extent of transitional-cell carcinoma of the urinary bladder. PATIENTS AND METHODS: Serum samples were collected from 113 patients with bladder cancer and from 200 healthy controls. Of the 113 patients, 59 had superficial bladder cancer and 54 had muscle-invasive cancer. Thirteen bladder cancer tissues (eight superficial and five muscle-invasive) were also collected. The levels of hepatocyte growth factor in the serum and tissues of these individuals were measured by enzyme-linked immunoadsorbent assay using hepatocyte growth factor antibodies. RESULTS: The levels of hepatocyte growth factor in the serum and tissues of patients with muscle-invasive cancer were significantly higher than those of patients with superficial bladder cancer (P <.0001 and P =.0054, respectively). The degree of elevation above the normal level of serum hepatocyte growth factor of the former (61.1%) was significantly higher than that of the latter (8.4%; P <.0001). The elevation was highest in patients with visceral metastasis (93.3%). Among patients with superficial bladder cancer, the overall survival rate of those with low levels of serum hepatocyte growth factor was significantly greater than that of those with high levels (P =.005). Among patients with minimally invasive bladder cancer, the disease-free and overall survival rates of those with high levels of serum hepatocyte growth factor were significantly lower than the same rates of those with low levels (P <.001 and P =.0028, respectively). CONCLUSION: Our study suggests that the level of hepatocyte growth factor in serum could be a predictor of patient survival and extent of bladder cancer.

Multifocal renal cell carcinoma: evidence for a common clonal origin.

The reported incidence of satellite tumor lesions in kidneys resected by radical nephrectomy for renal cell carcinoma (RCC) is 7-25%; however, genetic analyses of satellite tumors in comparison with those of main tumor lesions have not been performed well. In the present study, we investigated the incidence of loss of heterozygosity (LOH) at chromosome arms 3p, 6q, 8p, 9p, 9q, and 14q using 18 microsatellite markers in 10 nonpapillary RCCs of 50 mm or less in diameter and the accompanying satellite tumor lesions to evaluate the genetic alterations in main and satellite tumors. LOH was detected in 10, 3, 5, 3, 2, and 3 cases at chromosome arms 3p, 6q, 8p, 9p, 9q, and 14q, respectively. In addition, primary and satellite tumor lesions in 8 of 10 cases exhibited identical patterns of LOH on the 18 loci examined. In the remaining two cases, both main and satellite tumors demonstrated LOH on the common seven and three loci, respectively, whereas for another locus, LOH was observed only in the satellite tumor lesions. The similarity of LOH patterns detected in main and satellite tumor lesions indicates that the presence of satellite tumors might be the result of intrarenal metastasis from the main tumor lesion. These findings strongly suggest that even in case of small nonpapillary RCC, nephron-sparing surgery might carry the risk of failing to prevent postoperative local recurrence due to the incomplete resection of unrecognized satellite tumors with genetic alterations similar to those of the main tumor.

Relative expression of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 in mouse renal cell carcinoma cells regulates their metastatic potential.

To clarify the significance of the balance between matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) in the progression of renal cell carcinoma, we transfected both the MMP-2 and TIMP-2 genes simultaneously into RenCa, a mouse renal cell carcinoma cell line that does not express detectable levels of either MMP-2 or TIMP-2 mRNAs, and established several clones with various MMP-2:TIMP-2 expression ratios. On the basis of the quantitative evaluation of the MMP-2: TIMP-2 mRNA expression ratio by Northern blot analysis, we selected a clone overexpressing MMP-2 alone (RenCa/M), a clone overexpressing TIMP-2 alone (RenCa/T), and two kinds of clones overexpressing both, i.e., one with a high (RenCa/MTh) and one with a low (RenCa/MTl) MMP-2: TIMP-2 ratio, to compare the tumor cell phenotypes. In an in vitro tumor cell invasion assay, the MMP-2:TIMP-2 ratios of the RenCa sublines were directly correlated with their invasive potential. The invasive abilities of the parental RenCa cells induced by conditioned media from RenCa sublines were also correlated with the MMP-2:TIMP-2 ratios of the sublines. The cell adhesion assay showed the inverse correlation between the MMP-2 expression levels in the sublines and their cell adhesion to several extracellular matrix components. Furthermore, when injected i.v. or into the renal subcapsule in syngeneic mice, RenCa sublines formed metastatic nodules in the lungs, and the number of nodules was correlated with the MMP-2:TIMP-2 ratio of each clone. In contrast, despite the growth-inhibitory effects of TIMP-2 overexpression, MMP-2 overexpression had no effect on either proliferation in vitro of RenCa sublines or on their growth as tumors in vivo. These results suggest that the MMP-2:TIMP-2 expression ratio is a critical factor in the invasion and metastasis of renal cell carcinoma.

Epigenetic regulation of human bone morphogenetic protein 6 gene expression in prostate cancer.

Bone morphogenetic proteins (BMPs), belonging to the transforming growth factor-beta (TGF-beta) superfamily, are multifunctional molecules that regulate bone induction and organ development. Among BMPs, BMP-6 has been shown to be overexpressed in prostate cancer and is speculated to be associated with bone-forming skeletal metastasis. We investigated the regulatory mechanism of the BMP-6 gene expression in prostate cancer cell lines DU-145, LNCaP, PC-3, and PC-3M with regard to the methylation status of the CpG island in the 5' flanking region of the human BMP-6 gene. By sequence-specific analysis of methylated cytosines, we show here that the methylation status of the CpG loci around the Sp1 site of the BMP-6 promoter is related to its steady-state expression and an alternative splicing of messenger RNA (mRNA) in prostate cancer cell lines. Furthermore, a study of clinical cases of benign and malignant prostate lesion by in situ hybridization showed that BMP-6 expression was high at both primary and secondary sites in cases of advanced cancer with metastasis. Demethylation of the CpG loci around the Spl binding site was shown in cases with high BMP-6 expression by sequencing analysis of the methylated cytosine from paraffin-embedded materials. Our results suggested that during cancer progression, besides inactivation of tumor suppressor genes by hypermethylation, activation of certain genes like BMP-6 by selective demethylation was a common epigenetic event giving a variable character to the invading and metastasizing cancer cells.

Effectiveness of cancer vaccine therapy using cells transduced with the interleukin-12 gene combined with systemic interleukin-18 administration.

We introduced the interleukin-12 (IL-12) gene into mouse renal cell carcinoma (RenCa) cells to develop a tumor vaccine and to examine mechanisms of tumor rejection. IL-12-secreting RenCa (RenCa/IL-12) cells were completely rejected when implanted into syngeneic BALB/c but not athymic nude mice, suggesting that T cells were involved in this antitumor effect. Depletion of natural killer (NK) cells in nude mice did not affect the tumor growth of RenCa/IL-12. The simultaneous injection of mitomycin C-treated RenCa/IL-12 inhibited the tumor growth of parental RenCa injected at a distant site, whereas injection of mitomycin C-treated parental RenCa did not. The antitumor effect of RenCa/IL-12 as a cancer vaccine was induced by CD8+ T cells and NK cells and was inhibited by CD4+ T cells. Although the systemic administration of recombinant IL-18 (rIL-18) alone did not inhibit the tumor growth, it did enhance the cancer vaccine effect of RenCa/IL-12. The combination therapy of RenCa/IL-12 and the systemic administration of rIL-18 retarded even the growth of established tumors. The effector cells of this combination therapy consist not only of CD8+ T cells and NK cells but also of CD4+ T cells. This synergistic cancer vaccine effect of in situ secretion of IL-12 and the systemic administration of rIL-18 may be attributed to a functional change of CD4+ T cells.

Expression of basic fibroblast growth factor is associated with resistance to cisplatin in a human bladder cancer cell line.

To clarify the role of basic fibroblast growth factor (FGF-2) in the drug resistance of bladder cancer, we transfected the FGF-2 gene into HT1376, an FGF-2 negative human bladder cancer cell line. The FGF-2-transfected cell lines exhibited three- to four-fold higher resistant potential to cisplatin than the vector-only transfected control cell lines in vitro. When cisplatin was injected intraperitoneally after s.c. implantation of HT1376 sublines into nude mice, FGF-2 transfectants formed tumors about twice as large as did controls. In contrast, there was no significant difference in either cell proliferation in vitro or tumor growth in vivo among these cell lines without cisplatin treatment. Furthermore, DNA degradation following cisplatin treatment was markedly suppressed in FGF-2 transfectants compared to control cells. These results suggest that the expression of the FGF-2 gene plays an important role in the acquisition of the cisplatin-resistant phenotype of bladder cancer, probably through the protection against cisplatin-induced apoptosis.

Regulation of the ribosomal-RNA gene-expression in fibroblasts isolated from different human or murine tissues.

Ag-NOR stainings of the fibroblastic cultures derived from skin, lung and kidney of mice and humans have shown differential regulation of rRNA gene expression. Ag-NOR activities were found to be highest in the fibroblasts derived from the lung and the lowest in the skin-derived metaphases from both mice and humans and they were intermediate in the fibroblasts of kidney from both these species. Our observations indicate further that fibroblasts derived from different tissues are different not only in producing organ-specific growth factors but also in differential expression of ribosomal cistrons. This difference can also explain why orthotopic implantation of human tumor cells into nude mice is preferential compared with ectopic implantation in tumor take and metastasis.

Synergistic anti-tumor effect of 13-cRA and IFN-alpha/beta in mouse renal cell carcinoma.

The synergistic anti-tumor effect of 13-cis retinoic acid (13-cRA) and interferon-alpha/beta (IFN-alpha/beta) was investigated using a highly metastatic mouse renal cell carcinoma cell subline (RenCa/F). Although 13-cRA inhibited tumor growth in vivo as well as in vitro, IFN-alpha/beta did not. Combined administration of 13-cRA and IFN-alpha/beta significantly enhanced the anti-tumor effect of 13-cRA. The retinoic acid receptor (RAR)-alpha and RAR-gamma were expressed in RenCa/F cells. Treatment with IFN-alpha/beta did not influence the expression level of these receptors at the mRNA level, which suggests that the synergism of 13-cRA and IFN-alpha/beta is not mediated through the RAR.

p53 modulation of Fas/Apo-1 mediated apoptosis in a human renal cell carcinoma cell line.

The mutant p53 gene was transfected into ACHN, a wild-type p53-containing human renal cell carcinoma (RCC) cell line. The colony forming efficiency in soft agar in the mutant-type p53-transfected cell line (ACHN/MP) was significantly higher than that in the vector-only transfected control cell line (ACHN/C). The anti-Fas monoclonal antibody (CH11) induced apoptosis in the ACHN/C cells in a dose-dependent manner, whereas the effect of CH11 on the ACHN/ MP cells was markedly suppressed. In addition, the cytotoxic effect of CH11 on the ACHN/MP cells was augmented by the pretreatment with interferon- , but the corresponding effect on ACHN/C cells was not. These findings suggest that Fas-mediated therapy could be a novel approach to RCC, if interferon- treatment is added according to the p53 gene status.

Critical factors regulating site-specific brain metastasis of murine melanomas.

The intracarotid injection of B16 melanoma cells syngeneic to C57BL/6 mice and K-1735 melanoma cells syngeneic to C3H/HeN mice results in site-specific brain metastasis in C57BL/6 x C3H/HeN F1 mice. The K-1735 cells produce lesions only in the brain parenchyma, whereas the B16 cells produce lesions only in the meninges and ventricles. To determine the mechanisms that regulate this site-specific brain metastasis, we transfected the melanoma cells with DNA from plasmids pSV2neo or pSV2hvgro, which confer resistance to the drugs neomycin and hygromycin, respectively. Hybrids between the B16 and K-1735 cells were obtained by fusion. Cells of the K-1735 x K-1735 hybrid produced lesions only in the brain parenchyma of C57BL/6 x C3H/HeN F1 mice, whereas all B16 x K-1735 hybrids produced lesions only in the meninges and the ventricles. Initial cell arrest in the meninges or the brain parenchyma, production of collagenolytic activity, motility, and expression of CD44 did not predict or correlate with site-specific brain metastasis. The response of the different melanomas and hybrid cells to transforming growth factor-beta (TGF-beta) correlated with growth in the brain parenchyma. B16 cells and B16 x K-1735 hybrids bound more TGF-beta than K-1735 cells. The in vitro growth of B16 cells and all B16 x K-1735 hybrid cells was significantly inhibited by TGF-beta1 and TGF-beta2, whereas the growth of K-1735 cells and K-1735 x K-1735 hybrids was enhanced. Since TGF-beta is abundant in brain tissue, the results suggest that the ability of melanoma cells to proliferate in the brain parenchyma determines the production of site-specific brain metastasis.

The importance of orthotopic implantation to the isolation and biological characterization of a metastatic human clear cell renal-carcinoma in nude-mice.

We established a new human renal cell carcinoma system to study some properties of metastatic renal cancer cells and the influence of the organ environment on their metastatic potential. Renal cell carcinoma obtained from a surgical specimen was dissociated enzymatically. Cells were injected into the subcutis, kidney, cecal wall, and spleen of nude mice. Tumors grew in the subcutis and kidney. Only kidney tumors produced distant metastasis. Subcutaneous tumors were avascular and encapsulated, whereas kidney tumors were highly vascularized and invaded the kidney parenchyma. Cell lines were also established from several spontaneous lung metastases. The most metastatic cell line (LM-6) expressed higher levels of basic fibroblast growth factor, gelatinase, and urokinase activity. These results show that human neoplasms are heterogeneous for biologic properties, that orthotopic implantation is essential for the selection, growth, and metastasis of human renal cell carcinoma cells, and that metastatic cells must possess multiple properties to enable them to complete the process.

Malignant gonadal stromal tumor.

A case of malignant gonadal stromal tumor is reported, and the 24 cases reported in the English and Japanese literature are reviewed. A thirty-four-year-old male visited our hospital with a painless tumor in the left scrotum. Left high orchiectomy was performed under a diagnosis of testicular tumor, and histologic examination of the resected specimen demonstrated that it was a malignant gonadal stromal tumor. There has been no evidence of disease for forty-one months following surgery. Of the 15 cases reported in the English literature, retroperitoneal lymph node dissection was performed in 3 cases, and metastasis was present in the resected lymph nodes in 2 of the cases. These 3 patients survived with no evidence of disease for follow-up periods ranging between six months and five years. However, lymph node metastasis was observed in 5 of 6 patients who underwent orchiectomy alone or in combination with radiation to the retroperitoneal lymph nodes. The 3 patients described in the Japanese literature who had received high orchiectomy and retroperitoneal lymph node dissection survived with no evidence of disease for follow-up periods ranging from eighteen months to forty-one months following surgery. These findings indicate that retroperitoneal lymph node dissection is useful as a treatment for malignant gonadal stromal tumor. However, the prognosis for patients with advanced disease is very poor, because neither chemotherapy nor radiotherapy is effective. Corroborative study of many patients is necessary to understand the pathophysiology of malignant gonadal stromal tumor and to develop useful treatments.

Elevation of serum level of vascular endothelial growth factor as a new predictor of recurrence and disease progression in patients with superficial urothelial cancer.

OBJECTIVES: To determine whether the serum vascular endothelial growth factor (VEGF) level in patients with urothelial cancer could be used as a predictor of recurrence and disease progression. METHODS: Serum levels of VEGF in 51 healthy controls and 135 patients with urothelial cancer (81 superficial and 54 invasive cancers) were measured using a sandwich enzyme immunoassay, and the results were analyzed with respect to several clinicopathologic factors. RESULTS: Significant differences in the serum VEGF level were observed between healthy controls and patients with superficial urothelial cancer (34+/-12 pg/mL versus 49+/-27 pg/mL, P<0.001) and between healthy controls and patients with invasive cancer (34+/-12 pg/mL versus 51+/-35 pg/mL, P<0.001), whereas there was no significant difference in the serum VEGF level between superficial and invasive cancers (49+/-27 pg/mL versus 51+/-35 pg/mL, P = 0.31). Among patients with superficial cancer, the disease-free survival rate of patients with elevated serum levels of VEGF was significantly lower than that of patients with normal levels (P<0.05). The progression-free survival rate of superficial cancer patients with elevated serum levels of VEGF was also significantly lower than that of patients with normal levels (P<0.01). In addition, Cox's multivariate analysis revealed that the elevation of serum VEGF level was strongly associated with disease-free survival and progression-free survival in patients with superficial cancer (P<0.05). CONCLUSIONS: The results of this study suggest that the elevation of serum VEGF level could be used as a new independent predictor of recurrence and disease progression in patients with superficial urothelial cancer.

Expression of MAGE genes in testicular germ cell tumors.

OBJECTIVES: Some of the MAGE gene family that encode tumor-rejection antigens recognized by cytotoxic T lymphocytes are expressed at the mRNA level in various malignant tumors. However, these genes are silent in normal tissues, except in the testis and placenta. It is therefore important to understand how MAGE gene expression changes with malignant transformation of the testis. We investigated the expression of MAGE-1, -2, -3, and -4 genes at the mRNA level in testicular germ cell tumors. METHODS: Reverse transcriptase-polymerase chain reaction for MAGE genes was performed using 32 testicular germ cell tumor specimens. RESULTS: MAGE-1, -2, -3, and -4 mRNA was detected in 16 (72%), 15 (68%), 18 (82%), and 17 (77%) of 22 patients with pure seminoma or mixed type with seminomatous elements. MAGE-1 , -2, -3, and -4 mRNA was found in 2 (20%), 5 (50%), 4 (40%), and 4 (40%) of 10 patients with nonseminomatous germ cell tumor (NSGCT). The expression rate of MAGE-1, -3, and -4 mRNA was significantly higher in patients with seminomatous elements than that in those with NSGCT. MAGE expression did not correlate with disease progression. CONCLUSIONS: MAGE genes are more preserved in seminoma than in NSGCT. This suggests that seminoma has traits more similar to normal testis than does NSGCT.