Role of autophagy in oncolytic herpes simplex virus type 1-induced cell death in squamous cell carcinoma cells.

Herpes simplex virus type 1 (HSV-1) is one of the most widely studied viruses for oncolytic virotherapy. In squamous cell carcinoma (SCC) cells, the role of autophagy induced by neurovirulence gene-deficient HSV-1s in programmed cell death has not yet been elucidated. The oncolytic HSV-1 strain RH2, which lacks the γ34.5 gene and induces the fusion of human SCC cells, was used. RH2 replicated and induced cell death in SCC cells. RH2 infection was accompanied by the aggregation of microtubule-associated protein 1 light chain 3 (LC3) in the cytoplasm, the conversion of LC3-I to LC3-II and the formation of double-membrane vacuoles containing cell contents. No significant changes were observed in the expression of Bcl-2 or Bax, while a slight decrease was observed in that of Beclin 1. The autophagy inhibitors, 3-methyladenine (3-MA) and bafilomycin A1, did not affect viral replication, but significantly inhibited the cytotoxicity of RH2. The caspase-3 inhibitor z-DEVD-fmk and caspase-1 inhibitor z-YVAD-fmk also reduced the cytotoxicity of RH2. These results demonstrated that γ34.5 gene-deficient HSV-1 RH2 induced autophagic cell death in SCC cells as well as pyroptosis and apoptosis.

Enhancement of antitumor activity of herpes simplex virus gamma(1)34.5-deficient mutant for oral squamous cell carcinoma cells by hexamethylene bisacetamide.

Current oncolytic viruses exert only limited antitumor activity on their own. There is a need to increase their oncolytic capability. We evaluated the effect of a differentiating reagent, hexamethylene bisacetamide (HMBA), on the antitumor activity of a gamma(1)34.5-deficient herpes simplex virus type 1 (HSV-1) R849 for human oral squamous cell carcinoma (SCC) cells. Hexamethylene bisacetamide increased the viral yield, especially at a low input multiplicity of infection (MOI), and the transcription of immediate early genes of HSV-1. Hexamethylene bisacetamide treatment promoted the cytopathic effect of R849 and increased the proportion of dead cells. Hexamethylene bisacetamide produced more apoptotic cells in R849-infected cells as compared with parental HSV-1(F)-infected cells. The growth of oral SCC xenografts in nude mice was markedly suppressed by treatment with R849 in combination with HMBA, and the survival of the co-treated animals was significantly prolonged as compared with that of animals treated with R849 only. Herpes simplex virus type 1 mRNA was expressed in tumors and trigeminal neurons, but not in brain, lung, liver, and kidney. These results indicate that HMBA enhances the antitumor activity of R849 through the expression of immediate early genes without increasing its toxicity. Hexamethylene bisacetamide can be used as an enhancing agent for oncolytic therapy with HSV-1 mutants.

A modified repositioning system for segmental resection of the mandible.

Mandibular reconstruction is required after segmental resection of the mandible. Several techniques have been proposed but have several drawbacks. A modified system (based on Leibinger's titanium-positioning system) that can reposition the residual mandible easily and accurately without interfering with the reconstructive procedure was developed. This system has been used successfully in more than 10 patients, with no complications.

Expression of vasoactive intestinal polypeptide and amylase in a human parotid gland adenocarcinoma cell line in culture.

A neoplastic epithelial cell line initially established in culture from a human parotid gland adenocarcinoma grown in athymic nude mice with a BALB/c genetic background, which has an ultrastructure similar to that of the intercalated duct cell of the salivary glands, was examined for the expression of amylase and vasoactive intestinal polypeptide (VIP). The cultured cells were found by the peroxidase-antiperoxidase (PAP) method to express amylase and ultrastructurally to have secretory granules showing positive immunoreaction with anti-amylase serum. In addition, the cells were found to secrete amylase into the culture medium. The expression of VIP in the cultured cells was observed by the PAP method, immunofluorescent staining method, or immunoelectron microscopy. Moreover, the presence of the polypeptide reactive to antibodies directed against VIP in the cultured cells was confirmed by immunoblotting and radioimmunoassay. These findings indicate that the cells proliferating in culture express both amylase and VIP.

Enhanced replication of herpes simplex virus by hexamethylene bisacetamide.

Hexamethylene bisacetamide (HMBA) is a potent inducer of differentiation of tumor cells. The effect of HMBA on cell growth and replication of herpes simplex virus (HSV) was investigated in HEp-2 epidermal cells, IMR-32 neuronal cells, K562 myeloid cells, Daudi Burkitt lymphoma cells, and CCRF-CEM T-lymphoid cells. The growth of HEp-2 and IMR-32 cells was not affected by HMBA at concentrations from 0.5 through 2 mM. The growth of K562, Daudi, and CCRF-CEM cells was inhibited by HMBA at concentrations from 1 through 5 mM. When HSV-infected cells were incubated with 0.5 through 5 mM HMBA, a dose-dependent increase in virus yield was observed in HEp-2 and IMR-32 cells, but not in the other cell lines. These findings indicate that HMBA enhances the replication of HSV in epidermal and neuronal cells and that HMBA therapy may be responsible for the development of herpetic lesions.

Identification of epidermal growth factor as a component of the rat urinary bladder tumor-enhancing urinary fractions.

Using the heterotopically transplanted rat urinary bladder, we have shown that normal rat urine has a potent tumor-enhancing effect on bladder carcinogenesis. In an attempt to isolate tumor-enhancing factor(s), urine was fractionated by Bio-Gel P-100 column chromatography and each eluate fraction was examined for inducibility of ornithine decarboxylase (ODC) in a target rat bladder carcinoma cell line, 804G. We have identified two ODC-inducible peaks, one located in a high molecular weight region designated as Fraction I (Fr. I) and the second in a low molecular weight region designated as Fraction II (Fr. II). Fr. I consisted of two principal elements, transferrin and a component which induced ODC. The present investigation was conducted to characterize the ODC-inducible activity in Fr. I and II. Chromatographic analysis of Fr. I by Sephacryl S-200 and Fr. II by Bio-Gel P-10 chromatography separated several ODC-inducible peaks. However, the major ODC inducibility was due to a high concentration (460 ng/mg Fr. I residue, approximate Mr 54,000, and 580 ng/mg Fr. II residue, approximate Mr 6,100) of epidermal growth factor (EGF) as determined by radioimmunoassay. Aliquots obtained from these peaks competed with mouse EGF for EGF receptors in A431 cells. Preincubation of Fr. I and II with rabbit anti-rat EGF IgG significantly reduced ODC inducibility. Transforming growth factor alpha activity as determined by radioimmunoassay was also demonstrated in both Fr. I (34 ng/mg) and Fr. II (9 ng/mg). The results of the present study together with our previous data indicate that the majority of the ODC-inducing activity in the tumor-enhancing urinary components Fr. I and Fr. II is due to EGF itself and EGF-related growth factors of high molecular weight and that Fr. I also contains transferrin.

5-Azacytidine induction of stable myoepithelial and acinar cells from a human salivary intercalated duct cell clone.

A neoplastic human salivary intercalated duct cell clone was cultured in 5 microM 5-azacytidine for 5 days at 37 degrees C; then the cells were trypsinized and subcultured in growth medium without 5-azacytidine. Thereafter, subclones were cloned from the subculture. Of 12 subclones isolated, 7 clonal cell lines were established and characterized. The two subclones composed of cells which were spindle shaped or stellate exhibited phenotypes similar to those of myoepithelial cells such as microfibrils and myosin and formed a myoepithelioma upon transplantation of the cells into nude mice. The other five subclones were composed of polygonal cells with numerous secretory granules in their cytoplasm and containing amylase that seems to be specific to acinar cells; transplantation of these cells into nude mice resulted in production of acinic cell carcinoma. These findings indicate that a neoplastic human salivary intercalated duct cell is capable of at least bidirectional differentiation.

Induction of cells with phenotypic features of neuronal cells by treatment with dibutyryl cyclic adenosine 3',5'-monophosphate in a human parotid gland adenocarcinoma cell line in culture.

A human parotid gland adenocarcinoma cell line, with an intercalated duct cell phenotype of the salivary gland and expression of vasoactive intestinal polypeptide and amylase, was cultivated in the presence of dibutyryl cyclic adenosine 3',5'-monophosphate (dB-cAMP). Morphological changes occurred; cells formed long cytoplasmic processes densely packed with ample microfibrils, as well as microtubules, and grew in a netlike appearance. In addition, it has been found by the immunofluorescence staining technique, immunoblotting, or immunoelectron microscopy that the cells treated with dB-cAMP express neurofilaments, neuron-specific enolase, synaptophysin, and HNK-1 antigen, as well as the alpha- and beta-chains of tubulin, whereas these antigens are not detected in untreated cells. The expression of vasoactive intestinal polypeptide detected diffusely in the cytoplasm of untreated cells was restricted to the cell membranes during the cultivation of cells in the presence of dB-cAMP, while expression of amylase persisted in the treated cells in a fashion similar to that in untreated cells. Moreover, both anchorage-independent and anchorage-dependent growth of the cells was markedly suppressed in the presence of dB-cAMP. After removal of dB-cAMP from the culture, the treated cells returned rapidly to the phenotype and growth rate of the untreated cells. These findings indicate that reversible conversion into cells with phenotypic features of neuronal cells of a human parotid adenocarcinoma cell line occurs in growth medium containing dB-cAMP.

Emergence of differentiated subclones from a human salivary adenocarcinoma cell clone in culture after treatment with sodium butyrate.

A human salivary gland adenocarcinoma cell line, which has ultrastructure and biological markers specific to the intercalated duct cells of human salivary glands, was cultured in 5 mM sodium butyrate for 12 days; then the cells were trypsinized, subcultured for an additional 16 days, and then transferred to growth medium without sodium butyrate. Morphological changes appeared about 1 wk after return to growth medium without sodium butyrate; cells being spindle or stellate in shape appeared in the treated cells, whereas the untreated cells were polygonal in shape. This morphologically altered phenotype persists after more than 14 mo of culture in growth medium without sodium butyrate. Of 40 subclones isolated, 2 clonal cell lines were established from the subculture and characterized. The other 38 subclones were accompanied by cell death during the subcultures. The clonal lines exhibited a phenotype similar to myoepithelial cells such as myosin, beta-chain of S-100 protein, myofilaments, and oxytocin receptor in addition to decreased tumorigenicity and anchorage-independent growth. These findings indicate that commitment to differentiation into myoepithelial cells and conversion from malignant to normal phenotype occur in a human salivary gland adenocarcinoma cell line following the sodium butyrate treatment.

Intermediate-sized filaments and specific markers in a human salivary gland adenocarcinoma cell line and its nude mouse tumors.

The adenocarcinoma cell line HSG from human salivary gland, which proliferates in vitro or in nude mice, was examined by the immunoperoxidase method for the expression of three different types of intermediate-sized filaments (IFs) and of specific antigens such as carcinoembryonic antigen, S-100 protein, secretory component, lactoferrin, myosin, tropomyosin, and actin. The cultured HSG cells were found to express three different types of IFs defined by antibodies to keratin, vimentin, and desmin. In HSG cells proliferating in vitro at 34 degrees C and 37 degrees C but not at 39 degrees C, the expression of tropomyosin and carcinoembryonic antigen was observed, although myosin and S-100 protein were not detected. The expressions of actin, lactoferrin, and secretory component were restricted to cultured HSG cells at 39 degrees C and 37 degrees C, respectively. Transplantation of HSG cells into nude mice resulted in the establishment of a nude mouse system with malignant characteristics such as invasion and metastasis. The expression of IFs in the primary tumors was restricted to keratin and desmin IFs, whereas coexpression of keratin, vimentin, and desmin IFs was observed in some neoplastic cells present in the metastatic tumors in regional lymph nodes and lung. In addition, expression of actin, myosin, tropomyosin, and S-100 protein was found in the metastatic tumors, whereas myosin and S-100 protein were not detected in the primary tumors. Moreover, the metastatic tumors were almost occupied by the neoplastic cells with oncocytic changes, although oncocytic change was not found in the cultured HSG cells and their primary tumors.

Effect of hexamethylene bisacetamide and cyclosporin A on recovery of herpes simplex virus type 2 from the in vitro model of latency in a human neuroblastoma cell line.

The goal of the present work was to examine whether hexamethylene bisacetamide (HMBA) and cyclosporin A affect the recovery of herpes simplex virus type 2 (HSV-2) from an in vitro model of HSV-2 latency in human neuroblastoma cell line IMR-32. IMR-32 cells were infected with HSV-2 at a multiplicity of infection of 0.1 plaque-forming units/cell and were cultured at 40 degrees C for 14 days, resulting in the establishment of a model of HSV-2 latency in IMR-32 cells. When the cultivation temperature was shifted down from 40 to 37 degrees C, recovery of virus growth began to occur after an incubation period of 2 days. During the time of shift-down of the incubation temperature, the latently infected cells were further cultured at 37 degrees C in the presence or absence of 5 mM HMBA or 0.5 micrograms/ml cyclosporin A, which does not affect stability of HSV-2 nor proliferation of IMR-32 cells. Consequently, the rate of HSV-2 recovery from the latently infected cells cultured in the presence of 5 mM HMBA was significantly increased, as compared with the untreated controls. In addition, the DNA methylation level of the latently infected IMR-32 cells cultured in the presence of HMBA was significantly decreased when compared to the level in the untreated controls. On the other hand, the cultivation of the latently infected cells in the presence of 0.5 micrograms/ml cyclosporin A resulted in a significant decrease in the rate of HSV-2 recovery. These findings indicate that the recovery of HSV-2 from the model of latency in IMR-32 cells is enhanced by HMBA treatment, which induces a significant decrease of total genomic DNA methylation level, and is inhibited by cyclosporin A treatment.

Expression of epidermal growth factor and transforming growth factor-beta in human salivary gland adenocarcinoma cell line.

Neoplastic intercalated duct cells that originated from a human submandibular salivary gland release transforming growth factor and human epidermal growth factor into serum-free medium. The transforming growth factor with the soft agar colony-forming activity when assayed in the presence of mouse epidermal growth factor on normal rat kidney clone 49F indicator cells, but without mitogenic action to quiescent 3T3 cells, does not compete with epidermal growth factor for receptor binding, is heat and acid resistant but trypsin and dithiothreitol sensitive, and therefore is of the transforming growth factor-beta class. The immunoreactive human epidermal growth factor is detected by radioimmunoassay on certain fractions obtained by the Bio-Gel P-60 chromatography of neoplastic epithelial duct cells originated from a human submandibular salivary gland-conditioned medium and is biologically very similar to mouse epidermal growth factor. Moreover, the presence of human epidermal growth factor in cultured neoplastic epithelial duct cells originated from a human submandibular salivary gland is demonstrated by the peroxidase:antiperoxidase method.

Immunogenic cell death by oncolytic herpes simplex virus type 1 in squamous cell carcinoma cells.

Molecules essential for the induction of immunogenic cell death (ICD) are called damage-associated molecular patterns (DAMPs). The effects of oncolytic herpes simplex virus type 1 (HSV-1) on the production of DAMPs were examined in squamous cell carcinoma (SCC) cells. The cytopathic effects of HSV-1 RH2 were observed in mouse SCCVII cells infected at a high multiplicity of infection (MOI), and the amounts of viable cells were decreased. After being infected with RH2, ATP and high mobility group box 1 (HMGB1) were released extracellulary, while calreticulin (CRT) translocated to the cell membrane. A flow-cytometric analysis revealed an increase in the number of annexin-V and propidium iodide (PI)-stained cells; and the amount of cleaved poly (ADP-ribose) polymerase (PARP) was increased. The killing effect of RH2 was reduced by pan-caspase inhibitor z-VAD-fmk and the caspase-1 inhibitor z-YVAD-fmk, suggesting the involvement of apoptosis and pyroptosis. In C3H mice bearing synergic SCCVII tumors, the growth of tumors injected with the supernatant of RH2-infected cells was less than that of tumors injected with phosphate-buffered saline (PBS). These results indicate that oncolytic HSV-1 RH2 produces DAMPs from SCC cells to induce cell death. This may contribute to the enhancement of tumor immunity by oncolytic HSV-1.

Phase II study of a novel oral formation of 5-fluorouracil in combination with low-dose cisplatin as preoperative chemotherapy of oral squamous cell carcinoma.

TS-1 is a novel oral 5-fluorouracil containing tegaful (prodrug of 5-FU) and two biochemical modulators. These modulators feature effect-enhancing and adverse reaction-reducing activity. We investigated the histological response and toxicities of combination chemotherapy with TS- 1 and low-dose cisplatin and evaluated its usefulness as preoperative chemotherapy Forty-four newly diagnosed patients with stage Il-IV oral squamous cell carcinoma were enrolled in this study from February 2002 to April 2004. Patients were administered TS-1 80 mg/m2/day (days 1-14) and cisplatin 5 mg/m2/day (days 1-5 and 8-12) followed by radical surgery within 2 weeks. The histopathological effect of chemotherapy, which was a surrogate endpoint of this trial, was evaluated with surgical or biopsy specimens. The rate of histological antitumor effect was as follows: complete response (CR) 36.4%, partial response (PR) 25.0%, minor response (MR) 18.1% and no change (NC) 20.5%. The rate of histological response (CR + PR) was 61.4%. The CR rate of effective cases was 59.3%. The main toxicities occurred in bone marrow and the digestive tract. The incidence of severe toxicity such as grade 3 or 4 was 4.5% in anemia, 9% in leukocytopenia, 11.4% in neutropenia, 4.5% in thrombocytopenia and 2.3% in anorexia, diarrhea and urticaria. Most patients showed no toxicity or mild toxicities. TS- 1 with low-dose cisplatin has highly effective antitumor activity and mild toxicities. In particular, the CR rate was very high. It is suggested that this regimen is suitable for neoadjuvant chemotherapy. We expect that this chemotherapy will contribute to avoidance of surgery for small tumors (stages I and II) and will enable function-preserving surgery for advanced tumors.

Ultrasound as a method to enhance antitumor ability of oncolytic herpes simplex virus for head and neck cancer.

Low-intensity ultrasound is a useful method to enhance the delivery of drugs to target cells via a range of mechanisms including the transient formation of micropores in the cell membrane, a process known as sonoporation. The effect of ultrasound on oncolytic herpes simplex virus type-1 (HSV-1) infection in oral squamous cell carcinoma (SCC) was examined. Human SCC cell line SAS and oncolytic HSV-1 RH2, which was deficient in the neurovirulent γ134.5 gene and exhibited cell fusion actions, were used. Cells grown in multi-well plates were infected with HSV-1 and exposed to ultrasound in the presence or absence of microbubbles after an adsorption period. The number of plaques was significantly greater than that of the untreated control. SAS cells were inoculated subcutaneously into nude mice and tumors were produced. Tumors were injected with HSV-1 RH2 with or without microbubbles and then exposed to ultrasound through the covering skin. The amount of the virus in tumor tissues 3 days after the injection was higher in tumors treated with HSV-1 RH2 and ultrasound than in tumors treated with RH2 only. The expression of the HSV-1 antigen was also increased by ultrasound and microbubbles. Tumor growth was suppressed with HSV-1 RH2 in combination with ultrasound, especially with microbubbles. These results indicated that ultrasound increased the efficiency of the HSV-1 infection in SAS cells and nude mouse tumors. This method can potentially be useful to enhance the antitumor effects of oncolytic HSV-1 on head and neck cancer treatment.

Heparan sulfate as a mediator of herpes simplex virus binding to basement membrane.

Explants of human lip and oral mucosa were infected with herpes simplex virus (HSV) in vitro and the expression of viral antigen was investigated by immunofluorescent antibody staining. Viral antigen was demonstrated in the cells of basal cell layer and lower prickle cell layers. Moreover, an accumulation of viral antigen in the epithelial-mesenchymal junction was observed. To examine the possibility that the basement membrane has an affinity for HSV, the interaction between HSV and major basement membrane components including type IV collagen, laminin, fibronectin, and heparan sulfate was investigated. When tested by a plaque-reduction assay, only heparan sulfate inhibited HSV plaque formation by competing for the virus adsorption to HEp-2 cells. The inhibitory effects of heparan sulfate and heparin were not affected by pre-incubation of these glycosaminoglycans with antithrombin III, whereas de-N-sulfation resulted in a significant reduction of their inhibitory activity. These findings suggest that heparan sulfate is involved in the binding of HSV to the basement membrane and that N-sulfated glucosamine residues of heparan sulfate are essential for HSV binding. The basement membrane may act as a reservoir of HSV in muco-cutaneous tissues.

Expression of abnormal transcripts of the FHIT (fragile histidine triad) gene in ovarian carcinoma.

To elucidate the role of the FHIT (fragile histidine triad) gene in ovarian carcinogenesis, the expression of the gene was analysed by reverse transcription-polymerase chain reaction (RT-PCR) in 51 cases of ovarian carcinoma, 6 cases of borderline tumour and 4 cases of benign ovarian tumour. The concomitant expressions of normal and abnormal FHIT transcripts were detected in 39% of carcinomas and in 83% of borderline tumours, while benign tumours and normal ovarian tissues expressed only normal transcript. In addition, there were 4 (8%) cases of carcinoma lacking expression of normal FHIT transcript, all of which were in advanced stages (stage III-IV) and poorly differentiated. These results suggest that the expression of abnormal transcripts of the FHIT gene is a feature of ovarian malignant/borderline tumours and that the complete loss of normal FHIT expression is related to the progression of ovarian carcinoma in a subset of the cases. However, abnormal FHIT transcripts themselves were not associated with any clinicopathological parameters, such as clinical stage, histological subtype of tumour, grade of differentiation or outcome of the patient. Additionally, abnormal FHIT expression was not associated with the presence of loss of heterozygosity (LOH) at this locus, suggesting that abnormal FHIT transcripts are not derived from genetic alteration or that genetic alteration at this locus is complicated.

Combination effect of adenovirus-mediated pro-apoptotic bax gene transfer with cisplatin or paclitaxel treatment in ovarian cancer cell lines.

To develop a novel therapeutic strategy for ovarian cancer, we constructed a recombinant adenovirus which highly expresses pro-apoptotic Bax protein and examined its therapeutic effect on a series of ovarian cancer cell lines: A2780, A2780/cDDP, OVCAR-3 and SK-OV-3. A recombinant adenovirus carrying the Bax-alpha gene (AxCALNKYbax) induced high expression of the Bax-alpha protein in all the cell lines. The cytotoxic effect of Bax was observed in three ovarian cancer cell lines: the per cent reduction in the number of cells was 40.0% for cisplatin-sensitive A2780, 50.0% for cisplatin-resistant A2780/cDDP, and 64.8% for marginally cisplatin-resistant OVCAR-3. In contrast, it was only 12.3% for cisplatin-resistant SK-OV-3. Cisplatin-resistant A2780/cDDP had a p53 mutation and exhibited attenuated Bax induction after cisplatin treatment, which may explain why supplementation of Bax was effective in this chemoresistant ovarian cancer. Combination with cisplatin or paclitaxel enhanced the cytotoxic effect of Bax induction in all but one cell line including cisplatin-resistant A2780/cDDP. It appears that adenovirus-mediated Bax induction, with or without combination with conventional chemotherapy, useful strategy for the treatment of ovarian cancer.

Effects of urinary transferrin and ornithine decarboxylase-inducing fraction on rat bladder carcinogenesis.

Using heterotopically transplanted rat urinary bladder (HTB) system, we previously have shown that contact with urine enhanced bladder carcinogenesis initiated by carcinogen. In order to screen urine for promoter substances, several short term in vitro assays were developed and their results were correlated with the in vivo assay results. Chromatographically separated urine fractions were examined for the inability to induce ornithine decarboxylase (ODC), to enhance incorporation of [3H]thymidine in a bladder carcinoma cell line (804G) and to form colonies in soft agar by NRK-49F. Data from the ODC assay and soft agar colony formation correlated well with the results derived from chronic animal studies. Thus then two assays appear useful in further screening urine for promoter substance. Data furthermore indicate that ODC-inducing urine component(s) may play a primary role in the steps following initiation whereas transferrin, a mitogenic urine component, may play a secondary role.

Involvement of urine in epithelial-stromal interactions in urinary bladder carcinogenesis.

The role of urine in epithelial-stromal interactions in urinary bladder carcinogenesis was investigated using the 'Stroma' bladder model established in our laboratory. Rats treated with 0.05% N-butyl-N-(4-hydroxybutyl) nitrosamine (BHBN) in drinking water for 4 weeks or age-matched untreated rats served as donors of bladders with denuded epithelium ('stroma' bladders), which were resurfaced 4 days later with urothelial cells from rats treated with 0.05% BHBN for 4 weeks. Subsequently, the transplants received weekly injections of normal rat urine or saline. In the urine-free environment, cell implants developed hyperplastic changes but few tumor formations with no significant difference between the two types of 'stroma' bladder. In contrast, urine instillation stimulated neoplastic growth, and tumor-enhancing effect was significantly accelerated in the BHBN-treated 'stroma' bladder group as compared to the control group. These results suggest that epithelial-stromal interactions are altered in such a way that bladder carcinogenesis is enhanced by prior exposure of the bladder stroma to carcinogen and subsequent urine contact.