B-Cell chronic lymphocytic leukemia (B-CLL) / small lymphocytic lymphoma (SLL) consists of heterogeneous diseases that are distinguished by morphological, immunophenotypic and molecular features. MUM1 (multiple myeloma oncogene 1) is a protooncogene that is deregulated as a result of (6;14)(p25;q32) chromosomal translocation in multiple myeloma, and is also expressed in a variety of malignant lymphoma entities. We examined the expression of MUM1 in B-CLL / SLL, and found that 2 of 4 B-CLL-derived cell lines and 14 of 29 patients' specimens expressed MUM1 by immunohistochemical analysis. MUM1 expression was not associated with CD38 expression, somatic hypermutation of immunoglobulin heavy chain gene variable region (IgV(H)), or any other clinical characteristics of the patients. Interestingly, the patients who were positive for MUM1 showed shorter overall survival times than those who were negative for MUM1 (50% survival: 22 months vs. 82 months) (P = 0.0008, log-rank test). Multivariate analysis by Cox's proportional-hazards regression model showed that MUM1 expression and unmutated IgV(H) status were independent unfavorable prognostic factors in patients with B-CLL / SLL. These findings suggest that MUM1 expression is a useful prognostic factor in B-CLL / SLL. The biological role and mechanism of action of MUM1 in B-CLL / SLL need to be clarified for the development of therapies for patients with the poor prognostic subtype.
The involvement of two phenotypically different regulatory T cells in different stages of tumor growth was investigated. Treatment of BALB/c mice with anti-CD25 monoclonal antibody (mAb) (PC61), but not anti-CD4 mAb (GK1.5) before RL male 1 or Meth A inoculation caused tumor rejection. On the other hand, treatment of BALB/c mice with anti-CD4 mAb (GK1.5) but not anti-CD25 mAb (PC61) on day 6 after inoculation of the same tumors caused rejection. The findings suggest that CD4+CD25+ T cells downregulated the rejection response in the early stage of tumor growth. On the other hand, putative CD4+CD25- T cells downregulated the tumor rejection response in the late stage. Both CD4+CD25+ and putative CD4+CD25- T cells appeared to inhibit the efficient generation of cytotoxic T lymphocytes (CTL). The present study also demonstrated that the treatment of BALB/c mice with anti-CD25 mAb (PC61) at 4 or 6 weeks after 3-methylcholanthrene (3-MC) inoculation retarded tumor occurrence and prolonged survival.
This study was designed to establish an intrahepatic metastasis model to investigate the biology and therapy of hepatocellular carcinoma (HCC) in mice. A fragment of mouse HCC tumor CBO140C12 was orthotopically implanted into the mouse liver. The number of intrahepatic metastatic colonies and the volume of the implanted tumor increased in a time‐dependent manner. At 28 days after fragment implantation, all mice showed intrahepatic metastasis. Intravenous administrations of cisplatin and doxorubicin at 7 and 21 days after the implantation significantly sup‐ pressed the growth of the primary tumor nodule, but tended to inhibit intrahepatic metastasis. However, a marked decrease of body weight was observed during the experiment. On the other hand, an inhibitor of matrix metalloproteinases (MMPs), ONO–4817, decreased the gelatinase activity of MMP–9 secreted by CBO140C12 cells, and significantly reduced the number of colonies of intrahepatic metastasis when administered orally. Our established model, which is focused on intrahepatic metastasis, is suitable for evaluating the therapeutic effect of HCC and for analyzing intrahepatic metastasis, because this model reflects the clinical features of HCC and all the steps of tumor metastasis.
Recent reports showed that thalidomide has anti-angiogenic activity and is effective for the treatment of refractory multiple myeloma (MM). We examined the relationship between the clinical efficacy and adverse effects of thalidomide and the plasma concentrations of this drug as well as angiogenic growth factors in refractory MM. Ten out of twenty-four evaluable patients (42%) showed more than 25% reduction of M-protein, and eight (33%) achieved more than 50% reduction. These changes were associated with restoration of anemia and recovery of normal immunoglobulin level. Somnolence and headache, constipation, peripheral neuropathy and skin rash were frequently observed, but were well tolerated. However, grade 2 - 4 severe neutropenia was also observed in nine cases. These adverse effects other than neutropenia occurred more frequently in the patients with higher plasma concentrations of thalidomide (2.0 microg/ml at 12 h after the last administration) and were readily alleviated by dose reduction. In contrast, neutropenia developed regardless of the plasma concentration. Plasma concentrations of angiogenic growth factors were frequently elevated before treatment. After thalidomide treatment, these growth factor levels tend to decrease to near-normal ranges in responders but were still high in most non-responders. After thalidomide treatment, plasma vascular endothelial growth factor (VEGF) level was significantly reduced in responders (P = 0.025), but not in non-responders (P = 0.37). Reduction of plasma VEGF level might be an important indicator for anti-myeloma effect of thalidomide.
The stability and biological behavior of an in vitro system of doxorubicin (DXR) entrapped in NK911, polymer micelles, was examined and compared with those of DXR entrapped in Doxil, polyethylene-glycol-conjugated liposomes. The fluorescence of DXR inside micelles or liposomes in an aqueous solution is known to be strongly quenched by the outer shells of the micellar or liposomal formation. Thus, by measuring the fluorescence intensity of DXR released from NK911 or Doxil, we could determine the stability of the micellar or liposomal DXR formation. Furthermore, NK911 was found to be less stable than Doxil in saline solution. In drug distribution experiments using an in vitro solid tumor model, when spheroids formed from two human colonic cancer lines, HT-29 and WiDr, and a human stomach cancer line, MKN28, were exposed to NK911, DXR was distributed throughout the spheroids, including their center. On the other hand, when the spheroids were exposed to Doxil, DXR was distributed only to the surface of the spheroids. It has been suggested that Doxil can deliver DXR to a solid tumor more efficiently than NK911 via the EPR (enhanced permeability and retention) effect, because Doxil may be more stable in plasma than NK911. On the other hand, DXR packed in NK911 may be distributed by diffusion to cancer cells distant from the tumor vessel, because NK911 can leak out of the tumor vessel and may be able to release free DXR more easily than Doxil. It has been suggested that drug carrier systems such as liposomes and micelles should be selected appropriately bearing in mind the characteristics of the tumor vasculature and the tumor interstitium.
The precise mechanisms of resistance to camptothecin (CPT)-derived DNA topoisomerase (topo I) inhibitors and the determinants remain unclear. We found that a DNA repair protein, O(6)-methylguanine-DNA methyltransferase (MGMT), participated in resistance to irinotecan hydrochloride (CPT-11), its active metabolite SN-38, and a novel CPT derivative, DX-8951f. In 17 human cancer cell lines, MGMT gene expression level closely correlated with sensitivity to the CPT derivatives, and inhibition of MGMT activity by nontoxic 5 microM O(6)-benzylguanine augmented the drug activity in relation to the MGMT expression levels in 8 cell lines examined. Transfection of pCR / MGMT-sense into U-251MG and pCR / MGMT-antisense into T98G and HEC-46 cells revealed that increased MGMT expression decreased the sensitivity to CPT-11, SN-38, and DX-8951f, whereas repressed MGMT expression sensitized cells to the drugs. Western analysis revealed that treatment of MGMT-expressing T98G cells with the drugs caused a decrease of both MGMT and topo I in a dose-dependent manner. Although, in the transfectants, MGMT expression did not so closely correlate with the sensitivity to drugs as to nimustine hydrochloride (ACNU), MGMT is probably an important resistance determinant to CPT derivatives, and may play some role in the topo I-mediated DNA damage and / or the repair process.
We isolated a paclitaxel-resistant cell line (KK47/TX30) from a human bladder cancer cell line (KK47/WT) in order to investigate the mechanism of and reversal agents for paclitaxel resistance. KK47/TX30 cells exhibited 700-fold resistance to paclitaxel and cross-resistance to vinca alkaloids and topoisomerase II inhibitors. Tubulin polymerization assay showed no significant difference in the ratio of polymerized alpha- and beta-tubulin between KK47/WT and KK47/TX30 cells. Western blot analysis demonstrated overexpression of P-glycoprotein (P-gp) and lung resistance-related protein (LRP) in KK47/TX30 cells. Drug accumulation and efflux studies showed that the decreased paclitaxel accumulation in KK47/TX30 cells was due to enhanced paclitaxel efflux. Cell survival assay revealed that verapamil and cepharanthine, conventional P-gp modulators, could completely overcome paclitaxel resistance. To investigate whether new synthetic isoprenoids could overcome paclitaxel resistance, we synthesized 31 isoprenoids based on the structure of N-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine (SDB), which could reverse multidrug resistance (MDR), as shown previously. Among those examined, trans-N,N'-bis(3,4-dimethoxybenzyl)-N-solanesyl-1,2-diaminocyclohexane (N-5228) could completely reverse paclitaxel resistance in KK47/TX30 cells. N-5228 inhibited photoaffinity labeling of P-gp by [(3)H]azidopine, suggesting that N-5228 could bind to P-gp directly and could be a substrate of P-gp. Next, we investigated structural features of these 31 isoprenoids in order to determine the structural requirements for the reversal of P-gp-mediated paclitaxel resistance, suggesting that the following structural features are important for overcoming paclitaxel resistance: (1) a basic structure of 8 to 10 isoprene units, (2) a cyclohexane ring or benzene ring within the framework, (3) two cationic sites in close proximity to each other, and (4) a benzyl group with 3,4-dimethoxy functionalities, which have moderate electron-donating ability. These findings may provide valuable information for the development of P-gp-mediated MDR-reversing agents.
Uroplakins (UPs) Ia, Ib, II, and III are tissue‐specific and differentiation‐dependent transmem‐brane proteins of the urothelium. We assessed the usefulness of human UP Ia as a histological marker by examining its expression in urinary bladder transitional cell carcinoma (TCC). A polyclonal antibody against human UP Ia was raised using a synthesized polypeptide. We applied our antibody to various organ tissues, including urothelium, and observed no crossreactivity. Analysis by RT‐PCR of normal urothelium, TCC and other organ tissues indicated that the human UP Ia gene expression is highly specialized to urothelium, and is conserved in TCC. Using immunohistochemistry, we investigated the expression of UP Ia in TCC from patients who had undergone radical cystectomy and from autopsy cases. Positive staining (10% or more positive cancer cells) was noted in primary lesions from 61 of 63 (96.8%) cystectomy patients. Depending on pathological grade, high expression (50% or more positive cancer cells) was observed in 17 of 18 (94.4%) moderately‐ to well‐differentiated TCC and in 36 of 45 (80.0%) poorly differentiated TCC. With regard to tumor invasion, high expression was noted in 20 of 22 (90.9%) superficial and 33 of 41 (80.5%) muscle‐invasive TCC. Cause‐specific survival rates were 68.6% and 75.0% in high‐ and low‐expression patients, respectively (log‐rank test, P =0.855, mean follow‐up; 65.0 months). In metastases, positive reactions were observed in 13 of 18 (72.2%) lesions. UP Ia may represent a specific histological marker judging from the stable expression, although its value as a prognostic factor remains undetermined.
Resistance to chemotherapeutic drugs is one of the major difficulties encountered during cancer chemotherapy. To detect genomic aberrations underlying the acquired drug resistance, we examined three cultured human myelomonocytic leukemia cell sublines each resistant to adriamycin (ADR), 1‐β–1‐d‐arabinofuranosylcytosine (ara‐C), or vincristine (VCR), using comparative genomic hybridization (CGH), fluorescence in situ hybridization (FISH), RT‐PCR, and western blot techniques. Chromosomes 7, 10 and 16 most conspicuously showed frequent aberrations among the resistant sublines as compared to the parental KY–821 cell line. In ADR‐resistant cells, gains at 7q21, 16p12, 16p13.1–13.3, 16q11.1–q12.1, and losses at 7p22–pter, 7q36–qter, 10p12, 10p11.2–pter, 10q21–q25, 10q26–qter were notable. In ara‐C‐resistant cells, no remarkable gain or loss on chromosome 7, but losses at 10p14–pter, 10q26–qter and 16p11.2–p11.3 were observed. In VCR‐resistant cells, gain at 7q21 and losses at 10p11–p13, 10p15 and 16p11.2–p13.3 were found. FISH identified amplified signals for the MDR–1 gene located at 7q21.1 in ADR‐and VCR‐but not ara‐C‐resistant cells, and for the MRP–1 gene located at 16pl3.1 in ADR‐resistant cells. These findings were validated at the mRNA and protein levels. Overlapping of the amplified MRP–1 gene with MDR–1 gene may play a critical part in the acquisition of resistance to ADR. Resistance to ara‐C excluded MDR–1 gene involvement and highlighted other key genes such as MXR gene. Several other genes putatively involved in the development of drug resistance might lie in other aberrated chromosomal regions.
p185(HER-2/neu), a tyrosine kinase receptor, is one of the target molecules for cancer therapy, and its expression may reduce the sensitivity of tumor cells to anti-cancer drugs. p21(CIP1/WAF1) is a cyclin-dependent kinase inhibitor, and its expression may also be involved in chemoresistance. Non-small cell lung cancer (NSCLC) is a potentially systemic disease, and systemic therapies play an important role in its treatment. However, there have been no studies comparing the expression of these molecules between primary and metastatic tumors. We investigated the expression of p185(HER-2/neu) and p21(CIP1/WAF1) in 57 paired samples of primary NSCLC tumors and corresponding lymph node metastases by immunohistochemistry. Expression of each of p185(HER-2/neu) and p21(CIP1/WAF1) was highly correlated between primary tumors and lymph node metastases, and similar correlations were also obtained when adenocarcinoma and squamous cell carcinoma cases were analyzed individually. However we failed to detect any correlation between p185(HER-2/neu) and p21(CIP1/WAF1) expression. Our results suggested that expression of both p185(HER-2/neu) and p21(CIP1/WAF1) is concordant between primary and metastatic tumors.
We have sometimes encountered invasive ductal carcinomas (IDCs) of the pancreas containing intraductal carcinoma components in the intra‐ and/or extra‐tumor area. The purpose of this study was to investigate whether intraductal carcinoma components would be useful for predicting the outcome of IDC patients. Forty‐seven surgically treated IDCs were examined, and all histological tumor sections were stained with Elastica to accurately confirm intraductal carcinoma components. Well‐known clinicopathological parameters that exhibited a significant correlation in the univariate analyses for predicting disease‐free survival (DFS) and overall survival (OS) were entered into the Cox proportional hazard multivariate analysis. Since the lowest P ‐value predicting DFS or OS periods was observed in IDCs with more than 10% intraductal carcinoma components and those with 10% or less intraductal carcinoma components ( P =0.028 and P =0.019), we established the cutoff value of intraductal carcinoma components at 10%. In the multivariate analyses for DFS and OS, the presence of more than 10% intraductal carcinoma components showed a marginally significant increase in the hazard rate (HR) of tumor recurrence ( P =0.067) and significantly increased the HR of mortality ( P =0.040). The present study demonstrated that IDCs with more than 10% intraductal carcinoma components were associated with a significantly better patient outcome than those with 10% or less intraductal carcinoma components.
The drs gene was originally isolated as a transformation suppressor gene against the v‐src oncogene. Expression of drs mRNA is down‐regulated by retroviral oncogenes such as v‐src and v‐K‐ras in the rat cell line F2408. Expression of drs mRNA is also markedly reduced in a variety of human cancer cell lines, including those of carcinomas of the colon, bladder, and ovary, suggesting that down‐regulation of drs mRNA is correlated with the development of human cancers. To clarify the correlation between down‐regulation of the drs gene and malignant tumor formation in human colorectal neoplasms, we examined expression of drs mRNA in a variety of colon cancer tissues by in situ hybridization. A total of 53 morphologically distinct neoplastic specimens were divided into the following five groups according to morphology: low and high grade adenoma in 7 and 12 cases, respectively (groups A, B), protruded‐type carcinoma in 16 (group C), superficial‐type carcinoma with an adenomatous component in 10 (group D) or superficial‐type carcinomas without any adenomatous component in 8 (group E). Expression of drs mRNA was detected in normal mucosa, low‐grade adenoma and most superficial‐type carcinomas without any adenomatous component. On the other hand, the rate of drs mRNA expression was significantly lower in protruded‐type adenocarcinoma and superficial‐type carcinoma with an adenomatous component. Our results indicate that down‐regulation of drs mRNA is closely correlated with carcinomas which arise from adenomatous polyps in the course of the adenoma‐carcinoma sequence, but that most carcinomas arising de novo are independent of the tumor suppressor function of the drs gene.
The present study was designed to define molecular alterations in the initiation stage of rat stomach carcinogenesis. Groups of male Lewis rats, 6 weeks old, were given drinking water with or without N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG; 100 mg/liter). Total RNA was isolated from the stomach pyloric mucosa, and fluorescent differential display analysis was performed. A cDNA fragment of 125 bp encoding an extracellular matrix‐associated matricellular glycoprotein, osteonectin, was identified after 14 days of MNNG exposure. A severalfold increase in expression was observed after 14 and 27 days of MNNG exposure, as determined by northern blot and RT‐PCR. Immunohistochemistry revealed that osteonectin‐mAb‐stained flbroblastic cells appeared in interstitial tissue of pyloric mucosa. Additionally the gene expression of other extracellular matrix proteins, viz., collagen type III, fibronectin, osteopontin, proteoglycan NG2, laminin γ1 and S‐laminin, was also markedly increased, as determined by competitive RT‐PCR after 14 days of MNNG exposure. The gene expression of osteonectin and the six other extracellular matrix proteins was elevated in twelve stomach adenocarcinomas and adenomas induced by MNNG in Lewis and WKY rats. Osteonectin‐mAb‐stained flbroblastic cells were evident in interstitial tissue of stomach tumor. These results suggest that osteonectin‐expressing flbroblastic cells appear in the interstitial tissue of pyloric mucosa from the early initiation stage of rat stomach chemical carcinogenesis, and that this phenomenon probably plays a role in cancer development.
Alkylating agents or platinum analogues initiate several excision repair mechanisms, which involve incision of the DNA strand, excision of the damaged nucleotide, gap filling by DNA resynthesis, and rejoining by ligation. The previous study described that nucleotide excision repair permitted incorporation of fludarabine nucleoside (F‐ara‐A) into the repair patch, thereby inhibiting the DNA resynthesis. In the present study, to clarify the repair kinetics in view of the inhibition by F‐ara‐A, normal lymphocytes were stimulated to undergo nucleotide excision repair by ultraviolet C (UV) irradiation in the presence or absence of F‐ara‐A. The repair kinetics were determined as DNA single strand breaks resulting from the incision and the rejoining using the alkaline single cell gel electrophoresis (comet) assay. DNA resynthesis was evaluated in terms of the uptake of tritiated thymidine into DNA. The lymphocytes initiated the incision step maximally at 1 h, and completed the rejoining process within 4 h after UV exposure. UV also initiated thymidine uptake, which increased time‐dependently and reached a plateau at 4 h. A 2–h pre‐incubation with F‐ara‐A inhibited the repair in a concentration‐dependent manner, with the maximal inhibition by 5 μ M. This inhibitory effect was demonstrated by the reduction of the thymidine uptake and by the inhibition of the rejoining. A DNA polymerase inhibitor, aphidicolin, and a ribonucleotide reductase inhibitor, hydroxyurea, were not so inhibitory to the repair process as F‐ara‐A at equimolar concentrations. The present findings suggest that inhibition of nucleotide excision repair may represent a novel therapeutic strategy against cancer, especially in the context of resistant cells with an increased repair capacity.
A large number of human tumor antigens recognized by CD8+ cytotoxic T lymphocytes (CTL) have been identified. Some of them have been employed in clinical trials and have achieved some objective responses. However, little is known about those that are recognized by CD4+ T cells, except for a very few that were identified from melanomas. Previously, we reported that an oral squamous cell carcinoma (SCC) cell line, OSC–20, was effectively lysed by HLA‐DRB1·08032 (HLA‐DRS)‐restricted autologous CD4+ T cell line, TcOSC–20. In this study, we performed two steps of chromatographic purification of the tumor cell lysate in combination with mass spectrometry. We found one reverse‐phase high‐performance liquid chromatography (RP‐HPLC) fraction that was effectively recognized by the T cells. We analyzed the fraction by nano‐liquid chromatography/electrospray ionization ion trap mass spectrometry (LC/MS/MS) and found six representative ions. We could determine the primary amino acid sequence of each of the six ions. Three of them contained a potential HLA‐DR8 binding motif, and TcOSC–20 showed a rather strong cytotoxic response to one of the synthetic pep tides, namely, amino acid residues 321–336 of human a‐enolase. Thus, several gene products of squamous cancer cells are endogenously processed and may be presented on HLA class II molecules, so that they could constitute target molecules for autologous CD4+ T cells.
Recombinant viral vectors have been developed for use as therapeutic agents and for the introduction of exogenous genes into living cells. However, little is known about the viability and stability of such recombinant viruses during storage, transport and delivery under various conditions. We describe here an analysis of the stability of an adenoviral vector in crude solutions of cell lysates during freezing and thawing and during storage at various temperatures in the presence and in the absence of glycerol. For example, the titer of adenoviruses in crude lysates of infected cells was reduced only ten‐fold or three‐fold after two hundred rounds of freezing and thawing or after incubation at 28°C for 14 days, respectively. Our observations indicate that recombinant adenoviral vector was more stable than expected both during freezing and thawing and during storage at low temperatures. Our results confirm the importance of appropriate conditions for the delivery and transport of recombinant adenoviral vectors.
Natural killer (NK) cell neoplasms are rare diseases. Frequent abnormalities of the tumor suppressor genes Rb, p53, p15INK4B, p16INK4A and p14ARF have been reported. However, no oncogenes associated with tumorigenesis of NK cell neoplasms have been reported so far. We analyzed the status of oncogenes including N-ras, K-ras, H-ras, c-myc, N-myc and mdm2 by Southern blot, PCR-SSCP, western blot analysis and immunohistochemical staining. We analyzed four cell lines derived from NK cell neoplasms and 31 clinical samples with five subclasses of NK cell neoplasms. We found no point mutations of the ras family genes. We detected no mutations in the c-myc and N-myc genes. No overexpression of c-Myc protein was detected by western blot analysis. Although we found neither amplification nor rearrangement of the mdm2 gene, we found high expression of MDM2 protein in some cases by western blot analysis. Immunohistochemical staining confirmed the overexpression of MDM2 protein. We found 14 cases with overexpression of MDM2 protein out of 15 cases (93%) with four subclasses of NK cell neoplasms except chronic NK lymphocytosis. Our previous and these results suggested that the expression level of MDM2 protein is independent of the status of the p14ARF, p53, Rb genes. MDM2 protein might independently contribute to carcinogenesis of NK cell neoplasms. Although the number of the cases we analyzed was not large, alterations of ras and myc family genes may rarely contribute to tumorigenesis in NK cell neoplasms. In contrast, overexpression of MDM2 might be associated with tumorigenesis of NK cell neoplasms, especially aggressive subclasses.
Calponin h1 (CNh1) is a basic actin-binding protein that is abundantly expressed in smooth muscle cells and involved in smooth muscle contraction by inhibiting actomyosin MgATPase. In recent studies, CNh1 was noted to suppress cell proliferation and tumorigenicity in leiomyosarcoma and tumor growth in fibrosarcoma cell lines. To further investigate the function of CNh1 as a tumor suppressor, we transfected the human CNh1 gene into a v-src-transformed rat fibroblast cell line SR-3Y1 The volume of the tumors derived from one randomly selected CNh1-transfectant (C1) in nude mice was reduced to 34.1% of that from a randomly selected vector transfectant (V1). A similar tendency was observed in another independent pair (C2, V2). Pathological analysis showed a significant decrease in the number of mitotic cells in the CNh1-transfectants. Further, a marked reduction in the number of vessels in the CNh1-transfectant was observed. DNA synthesis under conditions without serum was significantly reduced in the CNh1-transfectant (C1) compared with the control transfectant (V1), while no significant difference was seen in the cellular growth in the presence of 10% serum. A slight but significant reduction in in vitro cellular motility in the CNh1-transfectant was also observed. While the suppression of growth potential and cell motility by CNh1 transfer was significant but partial, a marked reduction in vascular endothelial growth factor (VEGF) mRNA and the secretion of VEGF protein was observed in the CNh1-transfectant. These results suggest that CNh1 plays a role as tumor suppressor in SR-3Y1 mainly by decreasing VEGF expression and angiogenesis in vivo and partially through reducing cellular proliferative potential and cell motility.
The modifying effects of cigarette smoke (CS) exposure on a heterocyclic amine (HCA) 2‐amino‐3,8‐dimethylimidazo[4,5‐f]quinoxaline (MeIQx)‐induced carcinogenesis were investigated in male F344 rats. Groups 1 and 2 were fed MeIQx at a dose of 300 ppm, and simultaneously received CS and sham smoke (SS) for 16 weeks, respectively. Groups 3–5 were given the MeIQx diet for 4 weeks, and simultaneously exposed to CS for 4 weeks (group 3), exposed to CS for 12 weeks after the MeIQx treatment (group 4) or received SS for 16 weeks (group 5). Groups 6 and 7 were fed basal diet and respectively received CS and SS for 16 weeks. In terms of the mean number or area, the development of glutathione S‐transferase placental form‐positive (GST‐P+) liver cell foci was significantly (P<0.01) greater in group 1 than in group 2. The mean number of colonic aberrant crypt foci (ACFs) per animal was increased by continuous CS exposure regardless of MeIQx feeding, the differences between groups 4 and 5 (P<0.05), and between groups 6 and 7 (P<0.05) being significant. Immunoblot analysis confirmed that the hepatic CYP1A2 level in group 6 was remarkably increased as compared to that in group 7. In addition, liver S9 from rats in group 6 consistently increased the mutagenic activities of six HCAs including MeIQx as compared to those in group 7. Thus, our results clearly indicate that CS enhances hepatocarcinogenesis when given in the initiation phase via increasing intensity of metabolic activation for MeIQx and possibly colon carcinogenesis when given in the post‐initiation phase in rats induced by MeIQx.
Hypermethylation of the 5’upstream region (5’region) of the human p16CDKN2A (p16) gene is known to cause silencing, which is involved in a wide range of human cancers. For the rat p16 gene, its 5’region has not been cloned, and it is uncertain whether surrogate use of exon la is adequate for analysis of p16 silencing. In this study, we observed that methylation analysis of exon la gave false positive results in three samples of normal rat mammary epitheliums and in two of six primary mammary carcinomas. Therefore, we determined the nucleotide sequence of the 5’region of the rat p16 gene. To confirm that methylation status of the 5’region is correlated with p16 expression, the methylation status was analyzed by bisulfite sequencing and methylation‐specific PCR in three samples of normal mammary glands, six samples of mammary carcinomas and four cell lines. The 5’region was demethylated in all of the three normal and six carcinoma samples that fully expressed p16. On the other hand, the 5’region was highly methylated in the 3Y1 cell line, which lacked p16 expression, but without deletion. These results showed that the methylation status of the 5’region was more closely correlated with p16 expression than that of the exon 1α and analysis of the methylation status is useful in examining p16 silencing in various rat tumors.
