Epigenetic-genetic interactions in the APC/WNT, RAS/RAF, and P53 pathways in colorectal carcinoma.

PURPOSE: Early events in colorectal tumorigenesis include mutation of the adenomatous polyposis coli (APC) gene and epigenetic hypermethylation with transcriptional silencing of the O(6)-methylguanine DNA methyltransferase (MGMT), human mut L homologue 1 (hMLH1), and P16/CDKN2A genes. Epigenetic alterations affect genetic events: Loss of MGMT via hypermethylation reportedly predisposes to guanine-to-adenine or cytosine-to-thymine (G:C-->A:T) transition mutations in KRAS and P53, and silencing of hMLH1 leads to high levels of microsatellite instability (MSI-H)/mutator phenotype, suggesting that epigenetic-genetic subtypes exist. EXPERIMENTAL DESIGN: We evaluated the relationships of aberrant methylation of APC, MGMT, hMLH1, P16, N33, and five MINTs to mutations in APC, KRAS, BRAF, and P53 in 208 colorectal carcinomas. RESULTS: We found that APC hypermethylation was age related (P = 0.04), in contrast to the other genes, and did not cluster with CpG island methylator phenotype (CIMP) markers. Hypermethylation of APC concurrently with either MGMT or hMLH1 was strongly associated with occurrence of G-to-A transitions in APC [odds ratio (OR), 26.8; P < 0.0002 from multivariable logic regression model], but C-to-T transitions had no associations. There was no relationship of hypermethylation of any gene, including MGMT, with G-to-A or C-to-T transitions in KRAS or P53, although APC hypermethylation was associated with P53 mutation (P < 0.0002). CIMP with MSI-H due to hMLH1 hypermethylation, or CIMP with loss of MGMT expression in non-MSI-H tumors, was associated with BRAF mutation (OR, 4.5; P < 0.0002). CIMP was also associated with BRAF V600E T-to-A transversion (OR, 48.5; P < 0.0002). CONCLUSIONS: Our findings suggest that the heterogeneous epigenetic dysregulation of promoter methylation in various genes is interrelated with the occurrence of mutations, as manifested in epigenetic-genetic subgroups of tumors.

Hypermethylation status of APC inversely correlates with the presence of submucosal invasion in laterally spreading colorectal tumors.

Little is known about epigenetic alterations in laterally spreading colorectal tumors (LSTs). The goal of the present study was to elucidate the epigenetic background of LSTs and compare the methylation status of DNA CpG islands (CGIs) with clinicopathologic features. Methylation of MINT1, MINT2, MINT31, p16, O(6)-methylguanine-DNA methyltransferase (MGMT), adenomatous polyposis coli (APC), and human MutL homologue 1 (hMLH1) in 42 LSTs was assessed by methylation-specific polymerase chain reaction (MSP) and compared with clinicopathologic parameters. The frequency of hypermethylation was 12.5% (4/32) for MINT1, 40.0% (16/40) for MINT2, 25.0% (10/40) for MINT31, 25.7% (9/35) for p16, 7.7% (3/39) for hMLH1, 26.5% (9/34) for MGMT, and 35.9% (14/39) for APC. APC methylation was inversely associated with submucosal invasion (P = 0.034), which was not found in any of 14 LST cases with APC methylation, whereas submucosal invasion was present in 8 of 25 (32.0%) cases without APC methylation. These data suggest that hypermethylation of APC could be a predictive marker for the absence of submucosal invasion of LSTs.

Association of interleukin-19 gene polymorphisms with age.

We previously demonstrated a significant association of the interleukin-10 (IL-10) -819 T/C polymorphism with age. IL-19 stimulates the production of IL-10, and the IL-19 gene is located adjacent to the telomere side of the IL-10 gene. To explore the relationship between IL-19 single nucleotide polymorphisms (SNPs) and age, we genotyped 500 Japanese individuals (mean age: 56.7 years, range 19-100) for IL-19 Ser175Phe (T/C), -513 T/C, 1098 G/T (intron 1) and 5420 G/C (5'-untranslated region). Three of four SNPs (Ser175Phe, -513 T/C and 1098 G/T) exhibited a weak but significant association with age by chi-square test and logistic regression analysis (p <.05). IL-19 Ser175Phe was in linkage disequilibrium with -513 T/C and 1098 G/T, but not with IL-10 -819 T/C. These data suggest that IL-19 polymorphisms may be associated with age in a Japanese population.

Association of interleukin-10 promoter single nucleotide polymorphisms -819 T/C and -592 A/C with aging.

Increased inflammatory activity is known to accompany aging. Single nucleotide polymorphisms of inflammatory mediator genes might therefore affect the aging process. Relation of eight SNPs (tumor necrosis factor-alpha [TNF-alpha] -1031 T/C, interleukin-10 [IL-10] -819 T/C, IL-1beta -511 C/T, IL-6 -634 C/G, IL-18 -607 A/C, transforming growth factor-beta [TGF-beta] +869 C/T, matrix metalloproteinase-1 [MMP-1] -1607 1G/2G, and MMP-3 -1171 5A/6A) with age or gender was evaluated in 500 Japanese persons (mean age: 56.7 years old, range: 19-100) by the chi-square test. There was a significant association of IL-10 -819 T/C with age (p =.0026). The association remained significant after multivariate logistic regression analysis (odds ratio for an age interval for 1 year, 1.009; 95% CI, 1.002-1.016). Furthermore, the genotype distribution of IL-10 -819 T/C was completely consistent with that of -592 A/C. These data suggest that IL-10 -819 T/C and -592 A/C may be a promising candidate for an aging-related gene in a Japanese population.

MUC1 peptide vaccination in patients with advanced pancreas or biliary tract cancer.

BACKGROUND: To evaluate the immunogenicity of MUC1 peptide vaccine in advanced pancreatic and bile duct cancers, a phase I clinical trial was conducted. MATERIALS AND METHODS: A 100-mer MUC1 peptide consisting of the extracellular tandem repeat domain and incomplete Freund's adjuvant were subcutaneously administered to 6 pancreatic and 3 bile duct cancer patients at weeks 1, 3 and 5 and doses ranging from 300 to 3000 microg. Circulating intracytoplasmic cytokine-positive CD4+ T cells and anti-MUC1 IgG antibodies were measured before and after vaccination. RESULTS: There were no adverse events, except for mild reddening and swelling at the vaccination site. In 8 patients eligible for clinical evaluation, 7 had progressive disease and 1 stable disease with a tendency for increased circulating anti-MUC1 IgG antibody after vaccination. CONCLUSION: This phase I clinical trial revealed the safety of a vaccine containing 100-mer MUC1 peptides and incomplete Freund's adjuvant.

Association of matrix metalloproteinase (MMP)-1 promoter polymorphism with head and neck squamous cell carcinoma.

To explore the relation of the MMP-1 1G/2G and MMP-3 5A/6A promoter polymorphisms with head and neck squamous cell carcinoma (HNSCC), DNA specimens extracted from peripheral blood cells of HNSCC patients and healthy controls were genotyped. The frequency of the MMP-1 2G/2G genotype was significantly higher in HNSCC patients (n = 140) than in age- and sex-matched controls (n = 223) (P = 0.042; OR, 1.56). In the MMP-3 polymorphism, there was no significant difference in the genotype distribution between patients and controls. These data suggest that the MMP-1 promoter polymorphism may be associated with HNSCC.

Polymorphisms of the IL-1beta and IL-1beta-inducible genes in ulcerative colitis.

BACKGROUND: Ulcerative colitis (UC) is a chronic disorder of undetermined etiology, but a genetic predisposition to UC is well recognized. Among cytokines induced in UC, interleukin 1 (IL-1) appears to have a central role because of its immunological upregulatory and proinflammatory activities. The aim of this study was to assess whether UC is associated with polymorphisms of the IL-1beta gene and three additional genes inducible with IL-1beta in Japanese subjects. METHODS: A total of 96 patients with UC and 106 ethnically matched controls were genotyped at polymorphic sites in IL-1beta, matrix metalloproteinase 1 (MMP-1), matrix metalloproteinase 3 (MMP-3), and inducible nitric oxide synthase (iNOS) genes, using polymerase chain reaction (PCR)-based methods. RESULTS: There was no significant difference in genotype distributions of IL-1beta, MMP-1, MMP-3, and iNOS genes between controls and UC patients in a Japanese population. Also, no significant association of those polymorphisms with various clinical parameters of the patients was found. However, concerning association of age at onset with clinical factors in UC, the frequency of pancolitis was significantly higher in UC patients with age at onset being less than 30 years than in those more than 30 years of age (P = 0.049). CONCLUSIONS: No association of the IL-1beta and three IL-1beta-inducible gene polymorphisms with UC was observed in a Japanese population.

Increased expression of MUC1 in advanced pancreatic cancer.

BACKGROUND: MUC1 is associated with tumor invasion and metastasis, and is expressed in pancreatic cancer with a high frequency. This study explored whether MUC1 expression affected the survival of patients with pancreatic cancer. METHODS: Tissue specimens obtained from 70 patients with invasive ductal carcinoma of the pancreas, in pTNM stage III or IV, were immunostained with the anti-MUC1 monoclonal antibody DF3. The results of immunostaining were determined to be positive when more than 50% of the total cancer cells were positively stained. Association of the expression of the DF3 epitope with clinicopathological parameters or patients' survival was statistically evaluated. RESULTS: The incidence of positivity of MUC1 expression was 55.7% (39/70) and this incidence was significantly higher in pTNM stage IV than in stage III (odds ratio [OR], 4.015; 95% confidence interval [CI], 1.459-11.0541; P = 0.0076). As there was a clear difference in overall survival between pTNM stages III and IV ( P = 0.0016), the effect of MUC1 expression on survival was separately evaluated in each stage. It was shown that the expression of MUC1 was associated with unfavorable overall survival in stage IV ( P = 0.0197). CONCLUSIONS: Our data suggest that the expression of MUC1 may be related to the progression of pancreatic cancer.

Association of the -173 G/C polymorphism of the macrophage migration inhibitory factor gene with ulcerative colitis.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine and has been shown to be involved in the development of chronic murine colitis. In the +173 G/C polymorphism of the MIF gene, the presence of C creates the binding motif of activator protein 4. This study explored the association of this polymorphism with ulcerative colitis (UC). METHODS: Genotyping was carried out, with a tetra-primer polymerase chain reaction (PCR) method, for 659 DNA specimens from 438 healthy volunteers and 221 patients with UC. Genotype distribution between cases and controls and the association of patients' genotypes with clinical parameters were statistically evaluated. RESULTS: No significant difference in genotype distribution was found between UC patients and healthy controls. However, when the relation of the C/C genotype to clinical parameters in UC patients was evaluated by Fisher's exact test, it was found that the frequency of the C/C genotype was higher in patients with pancolitis type than in those with other types restricted to the distal or left-sided colon (odds ratio [OR], 10.781; 95% confidence interval [CI], 1.342-86.619; P = 0.0074). CONCLUSIONS: These data suggest that the MIF -173 G/C polymorphism may be related to the extent of disease in UC in a Japanese population.

Association of the OGG1 Ser326Cys polymorphism with tooth loss.

Although tooth loss is a serious health problem for elderly people, little is known about the genetic basis for susceptibility to it. In the present study we aimed to find a single nucleotide polymorphism (SNP) associated with tooth loss. DNA samples from 119 outpatients (mean age=78.8 years) were genotyped on seven polymorphisms (tumor necrosis factor-alpha -1031T/C, interleukin-1beta -511C/T, interleukin-6 -634C/G, macrophage migration inhibitory factor -173G/C, interleukin-1 receptor antagonist variable number of tandem repeat in intron 2, matrix metalloproteinase-1 -16071G/2G, and oxoguanine glycosylase 1 (OGG1) Ser326Cys (1245C/G)), and the results were statistically evaluated. Of the seven polymorphisms tested, only OGG1 Ser326Cys was revealed to associate with tooth loss at a statistically significant level (P=0.0086). In addition, a multivariate logistic regression analysis in which age, gender, body mass index (BMI), and ischemic heart disease were included as independent variables indicated that Ser326Cys could be an independent factor affecting tooth loss (OR, 3.191; 95%CI, 1.174-8.672). The data suggest that the OGG1 Ser326Cys polymorphism may be associated with tooth loss.

Anti-tumor activity of dendritic cells transfected with mRNA for receptor for hyaluronan-mediated motility is mediated by CD4+ T cells.

Receptor for hyaluronan-mediated motility (RHAMM) is overexpressed in various tumors with high frequency, and was recently identified as an immunogenic antigen by serologic screening of cDNA expression libraries. In this study, we explored whether RHAMM is a potential target for dendritic cell (DC) immunotherapy. We constructed a plasmid for transduction of in vitro-transcribed mRNAs into DCs to efficiently transport the intracellular protein RHAMM into MHC class II compartments by adding a late endosomal/lysosomal sorting signal to the RHAMM gene. Immunization of mice with modified RHAMM mRNA-transfected DCs (DC/RHAMM) induced killing activity against RHAMM-positive tumor cells in splenocytes. To examine whether CD4(+) and/or CD8(+) T cells were required for this antitumor immunity, an anti-CD4 or anti-CD8 antibody was administered to mice after immunization with DC/RHAMM. Depletion of CD4(+) T cells significantly diminished the induction of tumor cell-killing activity in splenocytes, whereas CD8(+) T cell depletion had no effect. We then investigated the therapeutic effect of DC/RHAMM in a 3-day tumor model of EL4. DC/RHAMM was administered to mice on days 3, 7 and 10 after EL4 tumor inoculation. The treatment markedly inhibited tumor growth compared to control DCs. Moreover, antibody-mediated depletion of CD4(+) T cells completely abrogated the therapeutic effect of DC/RHAMM, whereas depletion of CD8(+) T cells had no effect. The results of this preclinical study indicate that DCs transfected with a modified RHAMM mRNA targeted to MHC class II compartments can induce CD4(+) T cell-mediated antitumor activity in vivo.

Interaction of OGG1 Ser326Cys polymorphism with cigarette smoking in head and neck squamous cell carcinoma.

Recent molecular epidemiological studies have demonstrated that the human oxoguanine glycosylase 1 (OGG1) gene polymorphism may be associated with various cancers. To determine whether the OGG1 Ser326Cys polymorphism interacts with clinicopathological parameters including smoking and alcohol intake in head and neck squamous cell carcinoma (HNSCC), DNA samples from 192 patients with primary HNSCC were genotyped and studied by the case-only design. We observed an association between the Cys/Cys genotype and HNSCC with cigarette smoking of more than 40 pack-years by a multivariate logistic regression analysis (OR=8.10, 95% CI=1.06-61.73). No significant association of this genotype with alcohol intake was observed. Our present data suggest a possible interaction between the OGG1 Ser326Cys polymorphism and smoking in HNSCC.

Association of the BCRP C421A polymorphism with nonpapillary renal cell carcinoma.

Breast cancer resistance protein (BCRP), the second member of the ATP-binding cassette membrane transporter family, has a single nucleotide polymorphism, C421A (resulting in Q141K), that is of functional importance. Our aim was to explore the relationship between this polymorphism of the BCRP gene and the risk of renal cell carcinoma (RCC) development. For a case-control study, DNA samples from 200 nonpapillary RCC patients and 200 healthy control subjects were analyzed using the TaqMan technique. The genotypic frequencies of the BCRP C421A polymorphism were compared between RCC patients and control subjects. The frequency of the C/C genotype was significantly higher in RCC patients than in control subjects (age- and gender-adjusted OR = 1.96, 95% CI 1.32-2.93). No associations were observed between the BCRP C421A polymorphism and clinicopathologic or epidemiologic factors, including age, gender, tumor grade, stage, cigarette smoking, family history of cancer and body mass index. Carriers with the C/C genotype of the BCRP C421A polymorphism are at risk of developing nonpapillary RCC. These data suggest that BCRP is a candidate RCC susceptibility gene.

Circulating anti-MUC1 IgG antibodies as a favorable prognostic factor for pancreatic cancer.

MUC1 is immunogenic in vivo and humoral and cellular immune responses against MUC1 have been detected in cancer patients. Our study explored the association of circulating anti-MUC1 antibodies with clinicopathological parameters or patients' survival of pancreatic cancer. Serum specimens from 36 patients with invasive ductal carcinoma of the pancreas were subjected to enzyme immunoassay for anti-MUC1 IgG or IgM antibodies. Serum levels of anti-MUC1 IgG antibodies were significantly correlated with survival time (p = 0.0004), whereas neither those of anti-MUC1 IgM nor anti-Galalpha(1,3)Gal IgG antibodies, the latter known as natural antibodies cross-reactive with MUC1, showed a given tendency. Some patients' sera with the higher antibody titer showed the reactivity with MUC1-transfectants of cultured pancreatic cancer cells, but not with MUC1-negative parental cells. When the samples were tentatively divided into 2 groups by the serum level of anti-MUC1 IgG antibodies, the survival of patients was significantly longer in the group with optical density >or=0.3 than in that with optical density <0.3 (p = 0.008). Circulating anti-MUC1 IgG antibody levels remained significant (HR, 0.03; 95% CI, 0.003-0.289; p = 0.0024) after multivariate analysis for pTNM stage, patient age and gender. These data suggest that circulating anti-MUC1-IgG antibody levels may be predictive for survival of pancreatic cancer patients.

Evaluation of a new efficient procedure for single-nucleotide polymorphism genotyping: tetra-primer amplification refractory mutation system-polymerase chain reaction.

Tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) is a new efficient method for single-nucleotide polymorphism (SNP) genotyping. To determine the optimal conditions for ARMS-PCR we attempted to genotype ten SNPs. DNA was extracted from the peripheral blood of 168 unrelated healthy Japanese volunteers. Two problems inhibited uniform efficiency of the amplification of three bands. The first problem was the lower amplification efficiency of the shorter and allele-specific products compared with the largest product. This phenomenon was overcome by increasing the relative concentration of the inner primers. The second problem was non-specific amplification of the shorter products. To reduce the amplification of these non-specific bands, adjusting any one of the following PCR conditions was effective: i) reducing the ratio of the inner primer concentration relative to that of the outer primers; ii) increasing the annealing temperature for the initial 5-10 cycles; iii) hot start PCR. With these procedures all ten of the SNPs were successfully genotyped. Our present data may be useful in the further application of tetra-primer ARMS-PCR to SNP genotyping.

Evaluation of an mRNA lipofection procedure for human dendritic cells and induction of cytotoxic T lymphocytes against enhanced green fluorescence protein.

We utilized an mRNA lipofection procedure in human dendritic cells (DCs) and attempted to induce cytotoxic T lymphocytes (CTLs) against enhanced green fluorescence protein (EGFP). EGFP mRNA was transfected into phytohemagglutinin (PHA)-stimulated lymphocytes or adherent peripheral blood mononuclear cell-derived DCs using a liposomal reagent. Lipofection efficiency was measured by flow cytometry. In PHA-stimulated lymphocytes, increasing concentrations of liposome or mRNA increased EGFP expression levels by up to 64.4%, but caused a decrease in cell viability. A similar trend was also observed in DCs. For 70% DC viability, the concentration of liposomes was 24 microl/ml, and the mRNA concentration was 6 microg/ml. Under these conditions, ELISPOT and (51)Cr release assays were performed on CD8+ T cells stimulated twice with EGFP mRNA-transfected DCs. The number of interferon-gamma-producing cells was increased when the CD8+ T cells were cocultured for 24 h with PHA-stimulated lymphocytes transfected with EGFP mRNA. The level of specific lysis of EGFP mRNA-transfected DCs also increased to approximately 80%, with an effector to target ratio of 40:1. These data suggest that EGFP is immunogenic for human T cells, confirming that our lipofection procedure may be of use for inducing specific CTLs.

Association of a haplotype of matrix metalloproteinase (MMP)-1 and MMP-3 polymorphisms with renal cell carcinoma.

It has been shown that the matrix metalloproteinase (MMP)-1 promoter polymorphism 1G/2G is associated with an increased risk of developing various cancers including renal cell carcinoma (RCC), and is in linkage disequilibrium (LD) with the MMP-3 promoter polymorphism 5A/6A. These two genes are localized in 11q22 adjacent to each other. However, the relationship between the MMP-3 5A/6A polymorphism and susceptibility to cancer remains ambiguous. In this study, we genotyped eight polymorphisms in the region containing the MMP-1 and MMP-3 genes in 177 healthy subjects, and explored the relationships between RCC and these polymorphisms or haplotypes in 156 RCC cases and 230 age- and gender-matched controls. All the subjects studied were of Japanese descent. There were three polymorphisms that showed stronger LD with the MMP-1 1G/2G promoter variant than with the MMP-3 5A/6A promoter variant. One of these three polymorphisms was present in exon 2 of the MMP-3 gene and caused an amino acid change, Glu45Lys (G/A). When the genotype distribution of Glu45Lys was compared between RCC patients and controls, the frequency of the G/G genotype was significantly higher in the patients [age- and gender-adjusted odds ratio (OR) = 1.81, 95% confidence interval (CI) = 1.20-2.74]. A significant increase in the frequency of the 2G/2G genotype of the MMP-1 1G/2G polymorphism was also observed in the patients (age- and gender-adjusted OR = 1.86, CI = 1.23-2.82), whereas there was no significant difference for the MMP-3 5A/6A polymorphism. As expected based on these genotype-level results, the frequency of the 2G-G haplotype of MMP-1 1G/2G and MMP-3 Glu45Lys (G/A) polymorphisms was significantly higher in the patients than in the controls (crude OR = 1.95, CI = 1.31-2.91). These findings suggest that this haplotype of MMP-1 and MMP-3 variants may be associated with the risk of developing RCC.

Association of functional polymorphisms of matrix metalloproteinase (MMP)-1 and MMP-3 genes with colorectal cancer.

Matrix metalloproteinase (MMP)-1 and MMP-3 genes are associated with tumor cell invasion and metastasis with their promoter polymorphisms influencing the level of transcription. Our study explored the association of these polymorphisms with colorectal cancer risk in a Japanese population. DNA was extracted from peripheral blood of 101 patients with colorectal cancer and 127 age- and gender-matched healthy volunteers. Genotyping was carried out using PCR-RFLP and direct sequencing. In the MMP-1 gene polymorphism, the frequency of the 2G/2G genotype that is associated with higher enzyme activity was significantly increased in colorectal cancer patients when compared to controls (p = 0.0067; OR = 2.077; 95% CI = 1.221-3.534). With regard to the MMP-3 polymorphism, unexpectedly, the frequency of the 6A/6A genotype causing lower enzyme activity was significantly increased in patients (p = 0.0129; OR = 2.110; 95% CI = 1.165-3.822). Because the loci for the 2 MMP genes are closely linked, we examined linkage disequilibrium between the 2 loci using expectation-maximization algorithm. We found that the 2 loci were in linkage disequilibrium and that 2G-6A haplotype was significantly increased in patients compared to controls (p = 0.0010; OR = 1.949; 95% CI = 1.305-2.911). Our present data suggest that the MMP-1 and MMP-3 promoter polymorphisms may be associated with a colorectal cancer susceptibility in Japanese.

Therapeutic effect of dendritic cells loaded with a fusion mRNA encoding tyrosinase-related protein 2 and enhanced green fluorescence protein on B16 melanoma.

Dendritic cells (DCs) loaded with messenger RNA (mRNA) have been proposed to be useful for inducing specific cytotoxic T lymphocytes against tumor antigens. It is now also apparent that tumor antigen-specific T cell tolerance limits the efficacy of active immunotherapy. To improve the efficacy of mRNA-loaded DCs, we constructed a fusion mRNA encoding tyrosinase-related protein 2 (TRP2), which has a late endosomal/lysosomal sorting signal and enhanced green fluorescence protein (EGFP), and evaluated its effect in a murine melanoma model. C57BL/6 mice were challenged subcutaneously (s.c.) with 3 x 10(5) B16 tumor cells, and 3 and 10 days later, 3 x 10(5) DCs loaded with mRNA (DC/mRNA) were injected s.c. in the vicinity of the tumor site. Treatment with DC/TRP2 mRNA or DC/TRP2-EGFP mRNA significantly inhibited tumor growth compared to DC/PBS on day 17 after B16 challenge (DC/PBS vs. DC/TRP2 mRNA, p = 0.0411; DC/PBS vs.DC/TRP2-EGFP mRNA, p = 0.0253), whereas no antitumor effect was observed in mice treated with DC/EGFP mRNA or DC/TRP2 peptide. Moreover, the survival rate in mice immunized with DC/TRP2 mRNA or DC/TRP2-EGFP mRNA was significantly improved as compared with that in mice receiving DC/PBS (DC/PBS vs. DC/TRP2 mRNA, p = 0.0228; DC/PBS vs. DC/TRP2-EGFP mRNA, p = 0.0049). Depletion of CD4+ T cells or CD8+ T cells with antibody administration completely abrogated the therapeutic effectiveness of DC/TRP2-EGFP mRNA, suggesting the induction of a T cell immune response against the B16 tumor.