Carcinoembryonic antigen directed herpes viral oncolysis improves selectivity and activity in colorectal cancer

BACKGROUND: G207 is an oncolytic herpes virus whose replicative cycle requires cellular ribonucleotide reductase (RR) for viral DNA synthesis. We attempt to enhance viral cytotoxicity in carcinoembryonic antigen (CEA)-producing colorectal cancer (CRC) cells through CEA-driven RR production. METHODS: CEA enzyme-linked immunosorbent assay was performed on LS174T and HCT-8 human CRC cells. The CEA enhancer-promoter (CEA E-P) was functionally assessed by luciferase assay. CEA E-P was cloned upstream of UL39, the gene encoding the large subunit of RR. Cells were transfected with CEA E-P/UL39 and infected with G207 for cytotoxicity assays. LS174T, with or without CEA E-P/UL39, were implanted into athymic mouse flanks (n = 28) and treated with G207. RESULTS: CEA levels were 7-fold higher in LS174T versus HCT-8 ( P <.00001). CEA E-P increased luciferase expression 7.5-fold in LS174T ( P <.01), with no increase in HCT-8. G207 cytotoxicity of'CEA E-P/UL39-transfected LS174T cells increased 69% by day 10 versus nontransfected cells ( P <.001), with no significant increase in HCT-8. Combining CEA E-P/UL39 with G207 in LS174T flank tumors resulted in a 65% decrease in tumor volume versus G207, phosphate-buffered saline, or'CEA E-P/UL39 alone ( P <.0001). CONCLUSIONS: CEA-driven RR production by CEA-secreting CRC cells significantly improves oncolytic viral cytotoxicity and specificity in vitro, and reduces tumor burden in vivo.     

Utilizing tumor hypoxia to enhance oncolytic viral therapy in colorectal metastases

OBJECTIVE: To determine the effects of hypoxia-induced ribonucleotide reductase (RR) production on herpes oncolytic viral therapy. SUMMARY BACKGROUND DATA: Hypoxia is a common tumor condition correlated with therapeutic resistance and metastases. Attenuated viruses offer a unique cancer treatment by specifically infecting and lysing tumor cells. G207 is an oncolytic herpes virus deficient in RR, a rate-limiting enzyme for viral replication. METHODS: A multimerized hypoxia-responsive enhancer was constructed (10xHRE) and functionally tested by luciferase assay. 10xHRE was cloned upstream of UL39, the gene encoding the large subunit of RR (10xHRE-UL39). CT26 murine colorectal cancer cells were transfected with 10xHRE-UL39, incubated in hypoxia (1% O2) or normoxia (21% O2), and infected with G207 for cytotoxicity assays. CT26 liver metastases, with or without 10xHRE-UL39, were created in syngeneic Balb/C mice (n = 40). Livers were treated with G207 or saline. Tumors were assessed and stained immunohistochemically for G207. RESULTS: 10xHRE increased luciferase expression 33-fold in hypoxia versus controls (P < 0.001). In normoxia, 10xHRE-UL39 transfection did not improve G207 cytotoxicity. In hypoxia, G207 cytotoxicity increased 87% with 10xHRE-UL39 transfection versus nontransfected cells (P < 0.001). CT26 were resistant to G207 alone. Combining 10xHRE-UL39 with G207 resulted in a 66% decrease in tumor weights (P < 0.0001) and a 65% reduction in tumor nodules (P < 0.0001) versus G207 monotherapy. 10xHRE-UL39-transfected tumors demonstrated greater viral staining. CONCLUSIONS: Hypoxia-driven RR production significantly enhances viral cytotoxicity in vitro and reduces tumor burden in vivo. G207 combined with RR under hypoxic control is a promising treatment for colorectal cancer, which would otherwise be resistant to oncolytic herpes virus alone.     

Utilizing alpha-fetoprotein expression to enhance oncolytic viral therapy in hepatocellular carcinoma

OBJECTIVE: To determine whether alpha-fetoprotein (AFP)-regulated ribonucleotide reductase (RR) production would promote more vigorous and specific viral killing in AFP-expressing hepatocellular carcinoma (HCC). BACKGROUND: AFP is expressed in over 70% of primary HCC but not in normal adult liver. AFP production by HCC can be exploited to target viral killing of tumor cells. G207 is an oncolytic herpes virus lacking UL39, the gene encoding RR. RR is an enzyme required for viral DNA synthesis and cytotoxicity. METHODS: Enzyme-linked immunosorbent assay (ELISA) was performed for AFP levels on Hep3B and PLC5 human HCC cells. An AFP-albumin enhancer-promoter complex (AFP-alb) was constructed in a luciferase vector to assess function. AFP-alb was cloned upstream of UL39 (AFP-alb/UL39) and transfected into HCC cells for G207 cytotoxicity assays. Viral plaque forming assays evaluated G207 replication. Hep3B flank tumors, with and without AFP-alb/UL39 transfection, were established in athymic mice (n = 28) and treated with G207. RESULTS: Hep3B had 5-fold higher AFP levels than PLC5 (P < 0.00001). AFP-alb increased luciferase expression 72-fold in Hep3B (P < 0.001) and 3-fold in PLC5 (P < 0.001). AFP-alb/UL39 transfection increased G207 cytotoxicity 93% in Hep3B (P < 0.0005), with no significant increase in PLC5. Peak viral titers were 46-fold higher in Hep3B transfected with AFP-alb/UL39 versus mock-transfected cells (P < 0.01), with no significant change in PLC5. Flanks tumors transfected with AFP-alb/UL39 and treated with G207 demonstrated a 76% volume reduction versus mock-transfected tumors infected with G207 (P < 0.0001). CONCLUSIONS: AFP-driven RR production by hepatoma cells significantly enhances herpes viral cytotoxicity and specificity in vitro and reduces tumor burden in vivo.     

Paclitaxel enhanced radiation sensitization for the suppression of human prostate cancer tumor growth via a p53 independent pathway

BACKGROUND: This study investigated the influence of p53 status on treatment using combined paclitaxel and irradiation for human prostate cancer (PC) in vitro and in vivo. METHODS: Enhancement of the radiation response by paclitaxel was determined by MTT and clonogenic assays in four sublines of the human PC cell line, LNCaP, stably transfected to express different p53 mutations found in PC patients. Suppression of xenograft growth by combined paclitaxel and radiation was assessed in NOD.SCID mice in vivo. Expression of p53 and downstream functional proteins, p21 and Bax, was assessed by Western blotting. RESULTS: Paclitaxel (8-10 nM) suppressed cell proliferation by 50% by inducing G2M mitotic arrest in LNCaP cell lines transfected to overexpress wild-type or mutant p53. Exposure to 20 nM paclitaxel before radiation therapy enhanced cytotoxicity in clonogenic assays. The dose and duration of paclitaxel exposure were important in inducing both G2M arrest and cell growth suppression and were critical factors in paclitaxel/irradiation combination therapy. Western blotting indicated that combination therapy increased p21 protein expression to varying degrees in all cell lines. In vivo studies indicated that paclitaxel pre-treatment followed by irradiation significantly suppressed tumor growth compared with either treatment alone. CONCLUSIONS: Pre-treatment with paclitaxel enhances radiation efficacy on cell killing and suppression of growth of human PC cell lines in vitro and in vivo via p53 independent pathways. Paclitaxel has potential for use as a radiosensitizer in the treatment of patients with PC with either wild-type or mutant p53 genetic status.     

Effective treatment of pancreatic tumors with two multimutated herpes simplex oncolytic viruses

Pancreatic cancer is an aggressive, rapidly fatal disease against which current nonsurgical therapy has minimal impact. This study evaluates the efficacy of two novel, replication-competent, multimutated herpes viruses (G207 and NV1020) in an experimental model of pancreatic cancer. Four human pancreatic carcinoma cell lines were exposed to G207 or NV1020, and cell survival and viral progeny production were determined. Flank tumors in athymic mice were subjected to single or multiple injections of 1 X 10⁷ G207 or NV1020, and tumor volume was evaluated over time. For all of the cell lines, G207 and NV1020 produced infection, viral replication, and cell lysis (P <0.05). NV1020 resulted in a higher production of viral progeny compared to G207. The efficacy of viral tumor cell kill was greatest in those cells with the shortest in vitro doubling time. For flank tumors derived from hs766t, single or multiple injections of both viruses were equally effective and significantly reduced flank tumor burden (P <0.05). Complete hs766t flank tumor eradication was achieved in 25% (5 of 20) of animals treated with G207 and 40% (8 of 20) of animals treated with NV1020. In vivo efficacy correlated with in vivo tumor doubling time. There were no adverse effects related to viral administration observed in any animal. NV1020 and G2O7 effectively infect and kill human pancreatic cancer cells in vitro and in vivo. Given the lack of effective nonoperative treatments for pancreatic cancer, oncolytic herpes viruses should be considered for clinical evaluation. Presented in part at the Forty-First Annual Meeting of The Society for Surgery of the Alimentary Tract, San Diego, Calif., May 21–24, 2000.     

Treatment of human renal cell carcinoma by a conditionally replicating herpes vector G207

PURPOSE: Surgical removal remains the only potentially curative therapy for renal cell carcinoma. In this study we evaluated the inhibitory effect of the replication competent engineered herpes simplex virus type 1, G207, for renal cell carcinoma in vitro and in vivo. MATERIALS AND METHODS: The nature of G207 enables it to replicate within cancer cells, thus, causing cytolysis, but replication is restricted within normal cells. The susceptibility of the human renal cancer cell lines ACHN and A498 to G207 at a multiplicity of infection of 0.1 was examined. In addition, the growth characteristics of G207 was assessed. In vivo athymic mice bearing subcutaneous tumors were inoculated with 1 x 10(7) plaque forming units of G207 intra-neoplastically. For pathological analysis subcutaneous tumors were stained with X-gal. RESULTS: Two cell lines were efficiently destroyed by G207 within 1 week. The viral yields of G207 increased in a time dependent manner. In vivo the intra-neoplastic inoculation of G207 caused significantly decreased tumor growth in athymic mice harboring subcutaneous human renal cancer cells. On day 14 the mean growth ratio of ACHN and A498 lesions was significantly inhibited in G207 treated compared to control tumors (p <0.005 and <0.0001, respectively). CONCLUSIONS: These results suggest that G207 should be considered another potential therapeutic agent for renal cell carcinoma.     

Interleukin 12 secretion enhances antitumor efficacy of oncolytic herpes simplex viral therapy for colorectal cancer

OBJECTIVE: To assess the strategy of combining oncolytic herpes simplex virus (HSV) therapy with immunomodulatory therapy as treatment for experimental colon cancer. The oncolytic HSV recombinant NV1023 and the interleukin 12 (IL-12)-secreting oncolytic NV1042 virus were evaluated in vitro and in vivo with respect to antitumor efficacy. SUMMARY BACKGROUND DATA: Genetically engineered, replication-conditional, attenuated HSVs have shown oncolytic activity against a wide variety of solid malignancies. Other strategies for treating cancer have involved immunomodulation and cytokine gene transfer using viral vectors. This study has combined both of these strategies by inserting the murine IL-12 gene into a replication-competent HSV. This approach allows oncolytic therapy to replicate selectively within and lyse tumor cells while providing the host immune system with the cytokine stimulus necessary to recruit and activate inflammatory cells needed to enhance the antitumor effect. METHODS: NV1023 is a multimutant HSV based on the wild-type HSV-1 F strain. NV1042 was created by insertion of the mIL-12 gene into NV1023. Cytotoxicity and viral proliferation of both NV1023 and NV1042 within murine CT26 colorectal cancer cells were first shown. Cells infected with NV1042 were then shown to produce significant levels of IL-12. Using an experimental flank model of colon cancer, mice were treated with both high and low doses of NV1023 or NV1042 and were followed up for both cure and reduction in tumor burden. RESULTS: Both viruses could replicate within and kill CT26 cells in vitro, with 100% cytotoxicity achieved after infection by either virus. Only NV1042 could produce mIL-12. Therapy using high viral doses to treat animals in vivo showed equal efficacy between NV1023 and NV1042, with five of seven cures for each virus. When viral doses were lowered, only the cytokine-producing NV1042 virus could reduce tumor burden and cure animals of their disease. CONCLUSIONS: Both NV1023 and NV1042 have the oncolytic potential to kill colon cancer cells at higher doses. Cytokine production by NV1042 may allow lower doses of viral therapy to be used without losing antitumor efficacy. The combination of oncolytic viral therapy and immunomodulatory strategies should be further investigated as treatment for colon cancer.     

Oncolytic herpes viral therapy is effective in the treatment of hepatocellular carcinoma cell lines

The rising incidence of hepatocellular carcinoma (HCC) in western countries, along with the poor prognosis offered by present-day treatment modalities, makes novel therapies for this disease necessary. Oncolytic herpes simplex viruses (HSV) are replication-competent viruses that are highly effective in the treatment of a wide variety of experimental models of human malignancies. This study seeks to investigate the effectiveness of oncolytic herpes viruses in the treatment of primary HCC cell lines. Sixteen commercially available human HCC cell lines were studied. G207 is an attenuated, replication-competent, oncolytic HSV engineered to selectively replicate within cancer cells. Cell lines were tested for viral sensitivity to G207 and their ability to support viral replication using standard cytotoxicity and viral replication assays. Eleven of 16 cell lines were moderately to highly sensitive to G207 viral oncolysis. HCC cell lines additionally demonstrated the ability to support viral replication in vitro with as high as 800-fold amplification of the administered viral dose observed. G207 is cytotoxic to, and efficiently replicates within, HCC cell lines in vitro. From these data, we suggest that oncolytic HSV therapy may have a role in the treatment of HCC, and in vivo studies are warranted. Presented in part at the 2005 American Hepato-Pancreato-Biliary Association Congress, Hollywood, Florida, April 14–17, 2005. Supported by grants R01CA75461 and R01CA72632 from the National Institutes of Health, and by grant MBC-99366 from the American Cancer Society (Yuman Fong).     

The replication-competent oncolytic herpes simplex mutant virus NV1066 is effective in the treatment of esophageal cancer

BACKGROUND: The oncolytic herpes simplex-1 virus, NV1066, is a replication-competent virus that has been engineered to infect and lyse tumor cells selectively and to carry a transgene for enhanced green fluorescent protein (EGFP). The purpose of this study was to determine viral cytotoxicity in an esophageal cancer cell line and to determine whether EGFP expression could be used as a marker of viral infection. METHODS: BE3 esophageal adenocarcinoma cells were infected with NV1066 in vitro to determine cell kill and viral replication. EGFP expression was assessed by flow cytometry. The in vivo anti-tumor activity of NV1066 was tested in subcutaneous and intraperitoneal xenograft models. EGFP expression was localized in vivo by fluorescent microscopy and fluorescent laparoscopy. RESULTS: NV1066 effectively replicated within and killed BE3 cells in vitro and in vivo. EGFP expression identified infected tumor cells. After NV1066 treatment in vivo, EGFP expression localized to the tumor. In an intraperitoneal tumor model, EGFP could be visualized endoscopically using a laparoscope with a fluorescent filter. CONCLUSIONS: NV1066 has oncolytic activity against the BE3 cell line and may be a useful therapy against esophageal cancer. EGFP expression localizes the virus and may help to identify tumor deposits in vivo. Oncolytic activity with NV1066 against gastrointestinal cancers may potentially be tracked by endoscopy.     

Application of conditionally replicating herpes vector for gene therapy treatment of urologic neoplasms

Herpes vector has been widely used for experimental gene therapy. We herein review the strategies of such therapy for the treatment of urologic neoplasms. Most experimental studies of genetically altered viruses have employed replication-incompetent vectors. However, such viruses are unable to infect additional cells subsequent to the initial infection event. Therefore, this strategy has relied heavily on the bystander effect because a large number of noninfected tumor cells remain. Conditionally replicating herpes vector G207 has been developed in order to overcome potential problems of safety and tumor specificity for human use. It has been used to treat malignant brain tumors because of its neural tropism. In the last few years, applications of G207 for non-neural tumors have been reported. Because G207 may be useful for the treatment of urologic malignant tumors, we evaluated the antitumor effect against several types of tumor cells both in vitro and in vivo. Our data suggest that G207 may be applicable for the treatment of urologic malignant tumors.     

Antitumor activity of doxorubicin-monoclonal antibody conjugate on human bladder cancer

Doxorubicin (adriamycin) was conjugated via the dextran bridge method to a murine IgG3 monoclonal antibody, 1G3.10, directed against human bladder cancer. The drug-antibody conjugate, prepared from using 25% oxidized dextran as the linker, retained essentially the original immunological activity of the antibody using ELISA as tested against an antigen-positive target cell line (TSGH-8301), which has been shown to express an antigen recognized by the monoclonal antibody 1G3.10. Antitumor effect of the conjugate in vitro was evaluated by its inhibition on 3H-uridine incorporation into the established human bladder cancer cells. The conjugate exhibited a significantly higher cytotoxicity on target TSGH-8301 cells than that by a control antibody-doxorubicin conjugate prepared identically from an irrelevant mouse IgG3 monoclonal antibody. No apparently different cytotoxicity was detected on control antigen-negative bladder tumor cells of J82 between these two drug-antibody conjugates. Verapamil, a calcium channel blocker, enhanced the in vitro cytotoxicity of doxorubicin-1G3.10 monoclonal antibody conjugate. Results obtained from in vivo evaluation using xenografted target TSGH-8301 bladder tumor indicated that the 1G3.10 monoclonal antibody conjugate containing doxorubicin injected 4X, i.p., significantly inhibited TSGH-8301 bladder tumor growth in nude mice, whereas free monoclonal antibody, free drug and the mixture of both showed only moderate inhibition of tumor growth as compared to the untreated control. Verapamil also enhanced in vivo antitumor activity of the conjugate. There was no side effect (weight loss) detected on the conjugate-treated mice. Results obtained from in vivo evaluation using xenografted control J82 bladder tumor showed no specific antitumor activity as exhibited by doxorubicin-1G3.10 monoclonal antibody conjugate in comparison with free drug, mixture of drug and antibody without conjugation, or doxorubicin conjugated to the irrelevant antibody. These results suggested that doxorubicin conjugated with bladder tumor associated monoclonal antibody could be useful as a potentially cytotoxic agent in immunochemotherapy of human bladder cancer.     

Combination of genistein with ionizing radiation on androgen-independent prostate cancer cells

Aim: To study the effect of the combined use of genistein and ionizing radiation (IR) on prostate DUI45 cancer cells. Methods: DU145, an androgen-independent human prostate cancer cell line, was used in the experiment.Clonogenic assay was used to compare the survival of DU145 cells after treatments with genistein alone and in combination with graded IR. Apoptosis was assayed by DNA ladder and TUNEL stain. Cell cycle alterations were observed by flow cytometry and related protein expressions by immunoblotting. Results: Clonogenic assay demonstrated that genistein, even at low to medium concentrations, enhanced the radiosensitivity of DU 145 cells. Twenty four hours after treatment with IR and/or genistein, apoptosis was mainly seen with genistein at high concentrations and was minimally related to IR. At 72 h, apoptosis also occurred in treatment with lower concentration of genistein,especially when combined with IR. While both IR and genistein led to G2/M cell cycle arrest, combination of them further increased the DUI45 cells at G2/M phase. This G2/M arrest was largely maintained at 72 h, accompanied by increasing apoptosis and hyperdiploid cell population. Cell-cycle related protein analysis disclosed biphasic changes in cyclin B 1 and less dramatically cdc-2, but stably elevated p21^cip1 levels with increasing genistein concentrations.Conclusion: Genistein enhanced the radiosensitivity of DUI45 prostate cancer cells. The mechanisms might be involved in the increased apoptosis, prolonged cell cycle arrest and impaired damage repair. (Asian J Androl 2004 Dec; 6: 285-290)     

Enhancing the antitumoral effect of hypericin-mediated photodynamic therapy by hyperthermia

BACKGROUND AND OBJECTIVES: In the previous study, we have found a synergistic effect on the RIF-1 tumor cell killing when hypericin-mediated photodynamic therapy (PDT) was combined with hyperthermia. The purpose of the present study was to investigate the antitumoral effect of hypericin-PDT in combination with hyperthermia in the RIF-1 mouse tumor model. STUDY DESIGN/MATERIALS AND METHODS: Tumor response to PDT in combination with hyperthermia was compared to the response to PDT or hyperthermia alone. To explore the possible mechanism involved in the interaction of PDT and hyperthermia, we determined the tumor cell survival by in vivo/in vitro cell survival assay and analyzed the functional blood vessels by Hoechst 33342 staining. The mode of cell death was examined by TUNEL assay. RESULTS: Enhanced tumor response was obtained by PDT immediately followed by hyperthermia. Tumor cell survival assay revealed that indirect vascular effect contributed greatly to the overall tumor cell death induced by PDT with hypericin, whereas direct tumor cytotoxicity played a major role in hyperthermia-induced tumor cell killing. Combining PDT with hyperthermia brought about a synergistic interaction on direct tumor cell killing. Even though PDT or hyperthermia alone induced severe blood vessel shutdown and the combined treatments led to significant potentiation of the vascular damage as examined by Hoechst staining, the gain in tumor cell death as a result of this secondary vascular effect was limited after the combined treatments. Following the cellular damage by PDT in combination with hyperthermia, tumor cells were triggered to undergo apoptosis. CONCLUSIONS: Our study demonstrated the possibility of using hyperthermia to potentiate the antitumoral effect of hypericin-mediated PDT.     

Potent oncolytic activity of human enteroviruses against human prostate cancer

BACKGROUND: Oncolytic virotherapy offers a unique treatment modality for prostate cancer, especially stages that are resistant to current therapies, with the additional benefit of preferentially targeting tumor cells amongst an environment of healthy tissue. Herein, the low pathogenic enteroviruses; Coxsackievirus A21 (CVA21), as well as a bio-selected variant of Coxsackievirus A21 (CVA21-DAFv) and Echovirus 1 (EV1) are evaluated as novel oncolytic agents against human prostate cancer. METHODS: The surface expression of viral receptors required for enterovirus cell attachment/entry, including intercellular adhesion molecule-1 (ICAM-1), decay-accelerating factor (DAF) and integrin alpha(2)beta(1) on a number of human prostate cancer lines was assessed by flow cytometry. Susceptibility to viral oncolysis was determined via in vitro cell lysis assays performed on cell monolayers cultured in micro titer plates. The in vivo oncolytic efficacy of the enteroviruses was assessed using xenograft models in immune compromised SCID-mice following systemic challenge. RESULTS: The majority of prostate cancer lines tested expressed surface ICAM-1 and/or DAF, or alpha(2)beta(1), facilitating significant degrees of oncolysis following in vitro viral challenge. Systemic delivery of each of the three viruses induced reduction of xenograft tumor burdens in vivo, and a therapeutic dose-response was demonstrated for escalating doses of EV1 in the LNCaP animal model. CONCLUSION: Enteroviruses CVA21, CVA21-DAFv, and EV1 are potentially potent oncolytic agents against human prostate cancer.     

Structural analysis of the C-CAM1 molecule for its tumor suppression function in human prostate cancer.

BACKGROUND: Recently, we demonstrated that expression of C-CAM1, an immunoglobulin (Ig)-like cell adhesion molecule (CAM), was diminished in both prostate intraepithelial neoplasia and cancer lesions, indicating that loss of C-CAM1 expression may be involved in the early events of prostate carcinogenesis. Also, increased C-CAM1 expression can effectively inhibit the growth of prostate cancer. Structurally, C-CAM1 represents a unique CAM with a potential signal transducing capability. In this study, we further analyzed the functional domain of C-CAM1 for controlling its tumor suppression function. METHODS: Recombinant adenoviruses expressing a series of C-CAM1 mutants were generated, such as AdCAMF488 (mutated C-CAM1 containing Tyr-488 --> Phe-488), AdCAMH458 (intracellular domain deletion mutant containing 458 amino acids), AdCAMG454 (intracellular domain deletion mutant containing 454 amino acids), and AdCAMDeltaD1(C-CAM1 mutant containing first Ig domain deletion). After in vitro characterization of each virus, human prostate cancer cells infected with these viruses were subcutaneously injected into athymic mouse. Both tumor incidence and volume were measured for determining the tumor suppression function for each mutant. RESULTS: In vivo tumorigenic assay indicated that AdCAMDeltaD1 without cell adhesion function still retained its tumor suppression activity. In contrast, both AdCAMH458 and AdCAMG454 decreased or lost their tumor suppression activity. CONCLUSIONS: Our data indicate that the intracellular domain of the C-CAM1 molecule is critical for inhibiting the growth of prostate cancer, suggesting that C-CAM1 interactive protein(s) may dictate prostate carcinogenesis.     

Mutated ras p21 as a target for cancer therapy in mouse transitional cell carcinoma.

PURPOSE: To establish an experimental mouse model for bladder cancer immunotherapy using mutated ras as a target. MATERIALS AND METHODS: A tumorigenic mouse bladder transitional cell carcinoma (TCC) line MB49 (C57BL/6 origin) was analyzed for its c-ras gene status by DNA cloning and sequencing. Aberrant expression of the ras gene was measured with Western blotting. 13-mer peptides corresponding to residues 5 to 17 of the ras protein were synthesized and tested for immunogenicity in syngeneic C57BL/6 mice. Induction of specific immune responses was evaluated by analyzing splenocyte activity in vitro and tumor suppression in vivo. RESULTS: MB49 cells were found to contain a single amino acid substitution of serine for glycine at codon 12 in K-ras loci and an abundant amount of cellular mutated ras p21 protein. C57BL/6 mice immunized with the 13-mer serine-containing ras peptide exhibited mutation-specific immune responses in splenocyte proliferation, cytokine production and cytotoxicity. Specific antitumor immunity in the form of tumor growth delay in vivo was observed in mice immunized with the same mutant peptide followed by subcutaneous MB49 tumor challenge and was enhanced by the addition of low dose interleukin-12. CONCLUSIONS: The mouse bladder TCC line MB49 contains a serine mutation at codon 12 of its K-ras gene that is sufficient to induce mutation-specific immune responses in vitro and specific protective immunity to MB49 tumor in vivo. Mutated oncoproteins may be ideal targets for the development of specific immunotherapy regimens for bladder cancer immunotherapy.