转染hEndostatin基因对CNE2细胞株裸鼠移植瘤生长的影响

背景与目的：肿瘤的生长、转移是血管生成依赖性的,血管生成抑制剂有望通过抑制肿瘤血管生成及诱导肿瘤细胞凋亡,有效地抑制肿瘤的生长和转移。本文旨在探讨转染人内皮抑素基因hEndostatin对人鼻咽癌细胞株CNE2裸鼠移植瘤生长的影响。方法：采用脂质体介导法,分别将pBlast-hIL-hEndostatin、pBlast-hEndostatin和pBlast-MCS质粒导入人鼻咽癌细胞株CNE2,MTT法在体外检测转导细胞表达的hEndostatin的活性,并在体内实验中观察转导的CNE2细胞在裸鼠上的致瘤性的差异。结果：转染pBlast-hIL-hEndostatin的CNE2细胞的上清能显著抑制血管内皮细胞ECV304的生长,其在裸鼠体内形成的瘤组织重量和体积均显著低于对照组（P＜0．01）,生长速度明显慢于对照组细胞。结论：转染hEndostatin基因能有效抑制CNE2细胞在裸鼠体内的生长。     

SH2D1A expression in Burkitt lymphoma cells is restricted to EBV positive group I lines and is downregulated in parallel with immunoblastic transformation

The SH2 domain containing SH2D1A protein has been characterized in relation to the X-linked lymphoproliferative disease (XLP), a primary immunodeficiency that leads to serious clinical conditions after Epstein-Barr virus (EBV) infection. The SH2D1A gene is mutated in the majority of XLP patients. We previously detected SH2D1A in activated T and NK cells, but not in B lymphocytes. We have found SH2D1A protein in Burkitt lymphoma (BL) lines, but only in those that carried EBV and had a Group I (germinal center) phenotype. All the EBV-carrying Group III (immunoblastic) and the EBV-negative BL lines tested were SH2D1A-negative. Motivated by these differences, we studied the impact of EBV and the cellular phenotype on SH2D1A expression. We approached the former question with BL sublines after both the loss of the virus and subsequent reinfection. We also tested original EBV-negative BL lines carrying transfected EBV genes, such as EBNA1, EBNA2, EBNA6, EBER1, 2 and LMP1, respectively. In our experiments, no direct relationship could be seen between EBV and SH2D1A expression. We modified the phenotype of the Group I BL cells by LMP1 transfection or CD40 ligation. The phenotypic changes, indicated by expression of immunoblastic markers, e.g., SLAM, were accompanied by downregulation of SH2D1A. It seems, therefore, that the presence of EBV and the phenotype of the cell together regulate SH2D1A expression in the BL cells. It is possible that SH2D1A is expressed in a narrow window of B cell development represented by germinal center cells.     

Transfection by human oncogenes: concomitant induction of tumorigenicity and tumor-associated membrane alterations

NIH 3T3 cells were transfected with DNA derived from human bladder carcinoma, colon carcinoma and HL60 promyelocytic leukemia cells. The transfectants were examined for the presence of human oncogenes in relation to tumorigenic potential and composition of surface-located fucosyl glycopeptides by gel filtration, Concanavalin-A-binding and high-performance liquid chromatography. All transfectants, harboring 3 different human cellular ras genes, appeared to be tumorigenic in nude mice and displayed characteristically altered glycopeptides. The surface glycopeptides were consistently changed to higher apparent molecular weight due to enrichment in higher-branched sialic-acid-containing glycopeptides. Similar alterations have been found previously in virally- and chemically-transformed cells in vitro and tumors raised in vivo, and were designated as cancer-related or tumor-associated glycopeptides. Revertants derived from HL60-DNA-induced transfectants, which had lost the transfected human N-ras oncogene, simultaneously lost their tumorigenic potential and expression of cancer-related membrane glycopeptides. In addition, spontaneous transformants, exhibiting morphology and growth patterns indistinguishable from those of tumor-DNA-induced transfectants, neither contained transferred human DNA sequences nor expressed cancer-related glycopeptides. Nevertheless these cells were capable, after prolonged latency periods, of inducing tumors in nude mice. Cells derived from such tumors constantly displayed cancer-related glycopeptides on their surface, suggesting selection of tumorigenic cells from spontaneous transformants during passage in nude mice. In one of these tumors at least, an endogenous mouse ras-gene appeared to be activated. The results indicate a close correlation between the presence of activated ras-oncogenes in the genome of the transfected cells, the tumorigenic potential of these cells and the expression of surface-located cancer-related glycopeptides. The data suggest that functions provided by human ras-oncogenes contribute to the alteration of membrane glycopeptides on tumor cells.     

Burkitt's lymphoma cell lines reveal different degrees of tumorigenicity in nude mice

A quantitative in vivo assay for evaluating the tumorigenicity of Burkitt's lymphoma (BL) cell lines in nude mice is described. It is based on the dose-response kinetics of BL cell lines in pre-irradiated (480 rad) nude mice following the s.c. injection of 4 different cell doses. This model system was used to estimate the xenografting potential of 26 BL cell lines derived from BL patients of different geographic and ethnic origins, as well as lymphoblastoid cell lines (LCLs) established by Epstein-Barr virus (EBV) immortalization of normal B lymphocytes. The results indicate that most BL cell lines are tumorigenic, but LCLs fail to produce progressively growing tumours in nude mice. However, BL cell lines revealed individual degrees of tumorigenicity and accordingly could be divided into 4 groups with high, moderate, low or no tumorigenicity. Preliminary attempts to correlate the xenografting phenotype of BL lines with other characteristics indicate that: (1) the aberrations of chromosome 1 are more often encountered in cell lines with high and moderate tumorigenicity; (2) EBV-positive BL lines do not reveal a higher tumorigenic phenotype in comparison with EBV-negative ones; (3) cell lines carrying translocations t(8;22) and t(2;8) might fall more frequently in the group of lines with high and moderate tumorigenicity; and (4) when LCLs grow in nu/nu mice, rejection always occurs and is associated with massive tumour necrosis. These findings suggest that the tumorigenicity of BL cell lines in immunosuppressed animals is not related with EBV, but with certain chromosomal abnormalities (BL-specific and non-specific) indicating that this in vivo model system can be instrumental for the identification of other factors or stages involved in BL development.     

Analysis of the tumorigenic potential of common marmoset lymphoblastoid cells expressing a constitutively activated c-myc gene.

The respective roles of Epstein-Barr virus (EBV) and c-myc in the pathogenesis of endemic Burkitt''s lymphoma (BL) are unclear. In order to help resolve the question whether constitutive expression of the c-myc gene in an EBV-immortalised B cell is sufficient to induce a tumorigenic phenotype, B cells from a common marmoset (Callithrix jacchus) were immortalised with EBV, transfected with a constitutively activated c-myc gene and inoculated into the host animals. Despite the cell line transfected with c-myc displaying enhanced growth characteristics, in vitro and in vivo experiments demonstrated that this was not sufficient to induce a tumorigenic phenotype. This supports our previous findings with EBV-immortalised human B cells transfected with an activated c-myc gene (Hotchin et al., 1990).     

Transient expression of the Epstein-Barr virus LMP1 gene in human primary B cells induces cellular activation and DNA synthesis.

The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) and Epstein-Barr virus nuclear antigen 2 (EBNA2) are expressed in EBV-immortalized human B cells. It has previously been shown that transfection of the LMP1 and EBNA2 genes into Burkitt's lymphoma cell lines results in the up-regulation of CD23, CD21, ICAM-1 and LFA-1 cell-surface proteins. In the present study, the effects of transient expression of the LMP1 and EBNA2 genes were studied in normal primary human B cells pretreated with UV-inactivated EBV particles. To identify and purify cells which express the transfected DNA we used a gene encoding a surface molecule, CD2, as a co-transfection marker. We show that transient expression of the LMP1 gene, from heterologous promoters, is sufficient to induce cellular enlargement and up-regulation of surface expression of the activation markers CD23, CD21, ICAM-1 and LFA-1 in primary B cells. Most importantly, we show that transient expression of the LMP1 gene is sufficient to induce DNA synthesis in human primary B cells. Transient EBNA2 expression enhanced the effect of transient LMP1 expression on CD21 and CD23 cell-surface expression but, under our experimental conditions, inhibited the induction of DNA synthesis by LMP1. We conclude that activation of primary B cells with inactivated EBV particles, followed by transient expression of only two viral genes, EBNA2 and LMP1, is sufficient to reconstitute some of the early events of B-cell immortalization by EBV.     

Two Epstein-Barr virus (EBV) oncoproteins cooperate to repress expression of the proapoptotic tumour-suppressor Bim: clues to the pathogenesis of Burkitt's lymphoma

Epstein-Barr virus (EBV) contributes to the development of several human cancers including the endemic form of Burkitt's lymphoma (BL). In culture, EBV induces the continuous proliferation of primary B cells as lymphoblastoid cell lines (LCLs) and if EBV-negative BL-derived cells are infected with EBV, latency-associated viral factors confer resistance to various inducers of apoptosis. Nuclear proteins EBNA3A and EBNA3C (but not EBNA3B) are necessary to establish LCLs and their expression may be involved in the resistance of BL cells to cytotoxic agents. We have therefore created recombinant EBVs from which each of the EBNA3 genes has been independently deleted, and revertant viruses in which the genes have been re-introduced into the viral genome. Infection of EBV-negative BL cells with this panel of EBVs and challenge with various cytotoxic drugs showed that EBNA3A and EBNA3C cooperate as the main determinants of both drug resistance and the downregulation of the proapoptotic Bcl-2-family member Bcl-2-interacting mediator of cell death (Bim). The regulation of Bim is predominantly at the level of RNA, with little evidence of post-translational Bim stabilization by EBV. In the absence of Bim, EBNA3A and EBNA3C appear to provide no survival advantage. The level of Bim is a critical regulator of B cell survival and reduced expression is a major determinant of lymphoproliferative disease in mice and humans; moreover, Bim is uniquely important in the pathogenesis of BL. By targeting this tumour-suppressor for repression, EBV significantly increases the likelihood of B lymphomagenesis in general, and BL in particular. Our results may also explain the selection pressure that gives rise to a subset of BL that retain expression of the EBNA3 proteins.     

Hybridization between a human epithelial line, infectable by Epstein-Barr virus, and Burkitt lymphoma lines: membrane properties, superinfectability, inducibility and tumorigenicity

The human epithelial line U, which is partially infectable with EBV, was hybridized with the EBV-genome carrying Burkitt lymphoma lines P3HR-1 and Daudi. Authenticity of the hybrids U-Put and U-Dut was established by isoenzyme studies. Although the two hybrids carried the EBV genome derived from the lymphoma parent, being 100% positive for Epstein-Barr-virus-associated nuclear antigen (EBNA), they resembled the U parent in many respects: they were deficient for membrane immunoglobulins and Fc receptors, and had a lower concentration of EBV-C3 receptors than either parent. Unlike the P3HR-1 parent, U-Put hybrid was nonpermissive for both the EBV cycle antigens, early antigen (EA) and viral capsid antigen (VCA). The inducing agent 12-O-tetra-decanoyl-phorbol-13-acetate (TPA) caused distinct viral early antigen synthesis (EA) in U-Put, lower, however, than that of the parental P3HR-1. U-Dut was completely nonpermissive and noninducible for early and viral capsid antigens. Thus, even an epithelial parent infectable by EBV restricted, although not completely, expression of EBV antigens, with the exception of EBNA. It has been suggested that EBNA is an autonomous function of the viral genome, independent of host cell control; the latter regulates expression of antigens related to viral cycle. The hybrids U-Put and U-Dut resembled the U parent also in regard to growth in soft agar and tumorigenicity in nude mice, although in this respect the lymphoma parental properties were not completely eclipsed.     

Stable transfection of a human lymphoma line by sub-genomic fragments of Epstein-Barr virus DNA to measure humoral and cellular immunity to the corresponding proteins

An Epstein-Barr virus (EBV)-negative human lymphoid B-cell line, DG75, was stably transfected with recombinant selection vectors that carry a subfragment of the BamHI WYH region (nucleotides 44664 to 50628), the BamHI K fragment, or a subfragment of the EcoRI D region (nucleotides 166614 to 170149) of B95-8 EBV DNA. These fragments contain the coding exons for the EBV-determined nuclear antigens EBNA2 and EBNA1, and the membrane antigen LMP, respectively. Antigen expression of the cells was detected by immunofluorescence. EBNA2 was expressed in 80–100% of the transfected cells, in contrast to EBNA1 which was expressed in only 25%, and LMP in only about 5% of the cells. Humoral antibody responses were measured by immunofluorescence and compared to cellular immunity as determined by the leukocyte migration inhibition (LM1) technique. Extracts from transfected cell lines expressing EBNA1, EBNA2 or LMP elicited an LMI response with cells from healthy EBV-seropositive individuals whereas the extract from the parental DG75 cell line did not, The results demonstrate the value of stably transfected cell lines expressing a defined EBV antigen for the monospecific analysis of host responses to the EBV-encoded antigen complex in growth-transformed cells.     

Antiviral agent cidofovir decreases Epstein-Barr virus (EBV) oncoproteins and enhances the radiosensitivity in EBV-related malignancies

The Epstein-Barr virus (EBV) is involved in the carcinogenesis of several human cancers such as nasopharyngeal carcinoma (NPC) and Burkitt lymphoma (BL). Given the consistent role of EBV in transformation and maintenance of malignant phenotype, antiviral strategies provide an attractive approach to target EBV-expressing cells. In that aim, we have tested the Cidofovir, which is an acyclic nucleoside phosphonate analog known to exert an antiproliferative activity in some human virus-related tumors. Here, we show that Cidofovir induces a downregulation of the EBV oncoprotein LMP1 associated with a decrease of the antiapoptotic Bcl-2 and an increase of the proapoptotic Bax protein in Raji (BL) and C15 (NPC) cells. Using BL cell line BL2 B95-8 (BL2 infected with the B95.8 strain of EBV), we addressed the relation between EBV genome expression and modulation of viral oncoproteins by Cidofovir and/or ionizing radiation (IR). Cidofovir was able to significantly reduce LMP1 and EBNA2 mRNA and protein expression. This effect was associated with inhibition of proliferation, stimulation of apoptosis, and decrease of Bcl-2 expression in BL2 B95.8 cells. In addition, Cidofovir enhanced the radiation-induced apoptosis and the radiosensitivity through the proteolytic cleavage of death effectors caspase-9 and -3, which was specifically induced by combined treatment in EBV-positive cells compared to their negative counterparts. Furthermore, the combined treatment in nude mice led to a complete tumor remission without increasing toxicity in two human EBV-related cancer xenografts (Raji and C15). These results provide the basis for a novel anticancer strategy to enhance the therapeutic ratio of IR in EBV-related cancers.     

Correlation of growth capacity of cells in hard agarose with successful transfection by the activated c-Ha-ras oncogene and in vivo proliferative capacity at metastatic sites

The purpose of this study was to determine whether the degree of anchorage-independent growth of rodent or human cells in increasing concentrations of agarose correlated with successful transfection of the cells with an activated c-Ha-ras oncogene and tumorigenicity in nude mice. NIH 3T3 cells, C3H 10T1/2 fibroblasts, four clones of the murine K-1735 melanoma with different metastatic capacities and the TE85 human osteogenic sarcoma line were transfected with plasmids containing the 6.6-kilobase BamHI fragment of the mutant human c-Ha-ras gene and the neo gene, which confers resistance to neomycin (pSV2-neoEJ). Cells transfected with pSV2-neo, a plasmid containing the neo gene, served as controls. Cells from parental or transfected lines (selected by Geneticin) were plated into medium containing 0.3%, 0.6% 0.9%, or 1.2% agarose. These cells were also injected subcutaneously and intravenously into nude mice. The production of tumor cell colonies in dense agarose (greater than or equal to 0.6%) correlated with successful transfection with pSV2-neoEJ and production of experimental metastases in the lung of nude mice. We conclude that the degree of anchorage-independent growth of cells predicts successful transfection with activated c-Ha-ras oncogene and tumorigenic behavior in vivo. Thus this technique may be useful for the detection of cells transfected with transforming oncogenes.     

Transfer by pro gene transfection of tumor promoter-sensitive phenotype to promotion-insensitive JB6 cells

Transfection of activated promotion sensitivity genes (pro genes) confers on insensitive (P-) cells susceptibility to induction of anchorage-independent growth by tumor-promoting phorbol esters. Promotion-sensitive (P+) JB6 cell variants, from which activated pro-1 and pro-2 were cloned, respond to 12-O-tetradecanoylphorbol-13-acetate (TPA) and various nonphorbol tumor promoters with anchorage-independent transformation that is irreversible 60-80% of the time. Anchorage-independent (Tx) clonal lines derived from these TPA-induced agar colonies were also tumorigenic in nude mice. This report has addressed the question of whether the phenotypes associated with parental P+ cells are transferred by transfection of activated pro-1 and pro-2. Clonal lines were established after transfection of JB6 P- cells with activated pro-1 or pro-2, induction of anchorage-independent colony formation by TPA, and growth of individual agar colonies to yield clonal transfectant lines. The lines so derived from transfected populations included Tx, P+, and P- lines, reflecting irreversible neoplastic transformation and greater and lesser degrees of preneoplastic progression, respectively. The anchorage-independent transfectants were found to be tumorigenic. Since untransfected P- cells subjected to the same single-cycle TPA treatment and cloning in agar yielded no anchorage-independent and few P+ transfectants, the appearance of P+ and Tx transfectants after pro-1 and pro-2 transfection is therefore likely to be due to the transfected pro genes. Indirect assay of pro gene uptake by quick-blot hybridization of transfectant cell DNA with the vectors into which pro genes had been cloned confirmed the association of transferred P+ and Tx phenotypes with the presence of the transfected DNA. Finally, assay of the sensitivity of P+ pro-1 and pro-2 transfectants to transformation by TPA at various concentrations showed that transfection with pro-1 or pro-2 conferred about equal responses that were somewhat lower than those observed with parental P+ controls. Taken together these data indicate that promotion-insensitive JB6 cells need only an activated pro gene and TPA exposure to become neoplastically transformed.     

V-val突变型EB病毒核抗原1的功能研究

背景与目的:EB病毒核抗原1(Epstein-Barr viral nuclear antigen 1,EBNA 1)对于维持EB病毒的潜伏感染有重要作用。V-val变异型的EBNA1与鼻咽癌有密切的相关性。本研究旨在比较原型和V-val变异型的EBNA1基因在上皮细胞中的功能的差异。方法:用PCR方法扩增出原型和V-val变异型EBNA1基因的全长并克隆到pGFP载体上,转染HEK293细胞,检测两种亚型的EBNA1蛋白的表达对细胞生物学性状的影响。以含有EB病毒增强子FR序列的荧光素酶载体作为报告基因,比较两种亚型的EBNA1基因对质粒的转录激活能力。结果:原型和V-val变异型EBNA1基因的表达对HEK293细胞的生长速度没有明显影响,但表达原型EBNA1的细胞的克隆形成能力明显低于V-val亚型。在裸鼠致瘤实验中,接种表达原型和V-val变异型EBNA1细胞的实验组均未见肿瘤形成。但是在瞬时转染实验中,表达V-val变异型EBNA1基因的HEK293细胞的荧光素酶活性明显高于原型EBNA1基因。结论:原型和变异型EBNA1均未发现有明显的转化细胞的能力,但是V-val变异型EBNA1蛋白与原型相比,其转录激活的功能明显增强。     

The RON receptor tyrosine kinase is a potential therapeutic target in Burkitt lymphoma

Background: Aberrant expression of the RON receptor tyrosine kinase is associated with tumor progression and carcinogenesis. The aims of this study were to determine the role and functional mechanisms of RON in Burkitt lymphoma (BL) and to document its potential as a therapeutic target. Methods: RON expression was determined in BL cell lines by western blot analysis and examined in human lymphoma specimens by both western blotting and immunohistochemistry. The correlation between RON expression and Epstein-Barr virus (EBV) infection was investigated. Raji cells were treated with the Zt/f2 anti-RON mAb and cell viability, colony formation, apoptosis and cell cycle arrest were measured in vitro using cell proliferation assays, colony-forming assays and flow cytometry. Downregulation of RON by Zt/f2 was validated in mice bearing Raji cell xenografts.Results: Immunohistostaining showed a high frequency of RON⁺ cells in BL tissues and RON expression strongly correlated with EBV positivity. RON downregulation significantly decreased cell proliferation and colony formation via promotion of apoptosis and cell cycle arrest in Raji cells. The in vivo study showed that RON knockdown inhibits the tumorigenic potential of Raji cells in nude mice.Conclusions: RON acts as an oncogene in the carcinogenesis and progression of BL and is therefore a potential target for therapeutic intervention.     

Patients with Epstein Barr virus-positive lymphomas have decreased CD4(+) T-cell responses to the viral nuclear antigen 1

Epstein Barr virus (EBV) causes lymphomas in immune competent and, at increased frequencies, in immune compromised patients. In the presence of an intact immune system, EBV-associated lymphomas express in most cases only 3 or fewer EBV antigens at the protein level, always including the nuclear antigen 1 of EBV (EBNA1). EBNA1 is a prominent target for EBV-specific CD4(+) T cell and humoral immune responses in healthy EBV carriers. Here we demonstrate that patients with EBV-associated lymphomas, primarily Hodgkin's lymphoma, lack detectable EBNA1-specific CD4(+) T-cell responses and have slightly altered EBNA1-specific antibody titers at diagnosis. In contrast, the majority of EBV-negative lymphoma patients had detectable IFN-gamma expression and proliferation by CD4(+) T cells in response to EBNA1, and carry EBNA1-specific immunoglobulins at levels similar to healthy virus carriers. Other EBV antigens, which were not present in the tumors, were recognized in less EBV positive, than negative lymphoma patients, but detectable responses reached similar CD8(+) T cell frequencies in both cohorts. Patients with EBV-positive and -negative lymphomas did not differ in T-cell responses in influenza-specific CD4(+) T cell proliferation and in antibody titers against tetanus toxoid. These data suggest a selective loss of EBNA1-specific immune control in EBV-associated lymphoma patients, which should be targeted for immunotherapy of these malignancies.     

膜型基质金属蛋白酶-1上调人乳腺癌细胞血管内皮生长因子的表达并诱导肿瘤血管新生

目的 研究膜型基质金属蛋白酶-1(MT1-MMP)在肿瘤血管新生过程中的作用,探讨其诱导肿瘤血管新生的作用途径.方法 应用基因转染方法 ,将MT1-MMP导入人乳腺癌细胞系MCF-7细胞;应用半定量逆转录聚合酶链反应(RT-PCR)和免疫荧光染色,比较转染前后肿瘤细胞血管内皮生长因子(VEGF)表达的变化;通过裸鼠异种移植瘤模型,检测MT1-MMP对肿瘤生长速度、肿瘤组织微血管密度(MVD)和VEGF表达的影响.结果 在MT1-MMP稳定转染后的MCF-7细胞中,VEGF189、VEGF165和VEGF121 mRNA表达水平显著上调(P＜0.001).免疫荧光检测结果 显示,MT1-MMP组的VEGF蛋白免疫荧光强度为93.8±10.3,明显强于MCF-7组(42.9±5.3)和pcDNA3.1组(41.0±5.4,P＜0.001).裸鼠异种移植瘤模型结果 显示,MTI-MMP能够加快肿瘤生长速度.肿瘤组织MVD检测结果 显示,MT1-MMP能够显著提高肿瘤组织的MVD(P＜0.05).免疫组织化学染色结果 显示,VEGF在MT1-MMP组呈强阳性表达.结论 MT1-MMP能够通过上调肿瘤细胞VEGF表达水平而有效诱导肿瘤血管新生.MT1-MMP的这一作用途径可能为临床抗肿瘤研究及抗肿瘤药物开发提供新思路.     

Anti-tumor surveillance of non-contact-inhibited transformed cell lines

In order to determine if the correlated expression of transformation and tumorigenicity is affected by the agents used to induce transformants or by the immune status of the host used to test the tumorigenicity of transformants, we derived a series of cloned cell lines from foci of transformed cells induced by treatment of the contact-inhibited mouse cell line B/C-N7.ICI with the DNA demethylating agent 5-azacytidine (5-AZC) or the DNA demethylating and mutating agent benzo(a)pyrene dihydrodiol epoxide (BPDE). The transformed cell lines were injected into syngeneic nude and normal mice to determine their tumorigenicity. The results of this analysis showed that 93% of the transformants induced by 5-AZC treatment grew as tumors when injected into nude mice. Of those lines capable of growing as tumors in nude mice, 86% were also tumorigenic when injected into normal mice. In contrast, only 64% of BPDE-induced transformants grew as tumors in nude mice, and of those, only 44% were also tumorigenic in normal mice. The existence of non-contact-inhibited transformants that are tumorigenic only in nude mice indicates that host anti-tumor immune surveillance mechanisms are operative in normal mice. Further, the difference in both the percentage of transformed cell lines that are tumorigenic and the percentage of tumorigenic transformants that are susceptible to immune surveillance when transformants are induced by BPDE as compared to 5-AZC indicates that the transforming agent can affect both the correlation between the expression of transformation and tumorigenicity, and the interaction between the immune system and tumorigenic transformants.