ER-851, a Novel Selective Inhibitor of AXL, Overcomes Resistance to Antimitotic Drugs.

Innate and adaptive resistance to cancer therapies, such as chemotherapies, molecularly targeted therapies, and immune-modulating therapies, is a major issue in clinical practice. Subpopulations of tumor cells expressing the receptor tyrosine kinase AXL become enriched after treatment with antimitotic drugs, causing tumor relapse. Elevated AXL expression is closely associated with drug resistance in clinical samples, suggesting that AXL plays a pivotal role in drug resistance. Although several molecules with AXL inhibitory activity have been developed, none have sufficient activity and selectivity to be clinically effective when administered in combination with a cancer therapy. Here, we report a novel small molecule, ER-851, which is a potent and highly selective AXL inhibitor. To investigate resistance mechanisms and identify driving molecules, we conducted a comprehensive gene expression analysis of chemoresistant tumor cells in mouse xenograft models of genetically engineered human lung cancer and human triple-negative breast cancer. Consistent with the effect of AXL knockdown, cotreatment of ER-851 and antimitotic drugs produced an antitumor effect and prolonged relapse-free survival in the mouse xenograft model of human triple-negative breast cancer. Importantly, when orally administered to BALB/c mice, this compound did not induce retinal toxicity, a known side effect of chronic MER inhibition. Together, these data strongly suggest that AXL is a therapeutic target for overcoming drug resistance and that ER-851 is a promising candidate therapeutic agent for use against AXL-expressing antimitotic-resistant tumors.

Difference in the malignancy between RAS and GLI1-transformed astrocytes is associated with frequency of p27KIP1-positive cells in xenograft tissues.

We demonstrate that the introduction of GLI1 is sufficient for immortalized human astrocytes to be transformed whereas FOXM1 fails to induce malignant transformation, suggesting differences between GLI1 and FOXM1 in terms of transforming ability despite both transcription factors being overexpressed in malignant gliomas. Moreover, in investigations of mechanisms underlying relatively less-malignant features of GLI1-transformed astrocytes, we found that p27KIP1-positive cells were frequently observed in xenografts derived from GLI1-transformed astrocytes compared to those from RAS-transformed cells. As shRNA-mediated knockdown of p27KIP1 accelerates tumor progression of GLI1-transformed astrocytes, downregulation of p27KIP1 contributes to malignant features of transformed astrocytes. We propose that the models using immortalized/transformed astrocytes are useful to identify the minimal and most crucial set of changes required for glioma formation.

Xenografts Derived From Patients' Ascites Recapitulate the Gemcitabine Resistance Observed in Pancreatic Cancer Patients.

OBJECTIVES: Most patient-derived pancreatic ductal adenocarcinoma (PDAC) xenografts have been established from surgical specimens of patients who have not received chemotherapy. However, xenografts have rarely been established from chemotherapy-resistant, advanced PDACs, because such cases are usually inoperable. The purpose of this study is to establish patient-derived xenografts using PDAC cells refractory to chemotherapy. METHODS: Clinical PDAC cells obtained from ascites of patients who had received continuous chemotherapy were implanted into the flanks of immunocompromised mice. Growth and histological features of the xenografts with and without gemcitabine treatment were then analyzed. RESULTS: Ascites-derived PDAC cells were successfully expanded through serial xenograft passage without changes in histological appearance. While treatment with gemcitabine substantially inhibited the growth of all PDAC xenografts tested, the tumor volume gradually increased, and the tumors showed marked regrowth even under continued gemcitabine treatment. These findings are consistent with the actual clinical course of the corresponding patients for each xenograft. CONCLUSIONS: Ascites-derived xenograft models represent a valuable experimental system for testing the efficacy of currently available therapeutic compounds on chemotherapy-resistant PDAC cells and for elucidation of the mechanisms underlying chemotherapy resistance.

Imaging Window Device for Subcutaneous Implantation Tumor.

Most of preclinical cancer studies use xenograft models established from human cell lines or patient-derived cancer cells subcutaneously implanted into the flank of immunocompromised mice. These models are often assumed to represent the original diseases and are valuable tools, at least to some extent, for understanding both the basic biology of cancer and for proof-of-concept studies of molecularly targeted therapies. However, analyzing the cellular behavior of individual components within xenografts, including tumor cells, stromal cells, immune cells, and blood vessels, is challenging. In particular, it has been difficult and urgently required to trace the whole process of heterogeneous tumor microenvironment formation mediated by various components described above. Here we demonstrate a method for monitoring this process using a window device system that we have recently developed and a subcutaneous xenograft model that accurately recapitulates the histology of human lung adenocarcinoma. Use of our imaging window device and a multiphoton laser scanning microscope provides a powerful tool for investigating tumor heterogeneity and responses to drug treatments in an in vivo live imaging system.

Engineering cancer stem-like cells from normal human lung epithelial cells.

It has been proposed that a subpopulation of tumour cells with stem cell-like characteristics, known as cancer stem cells (CSCs), drives tumour initiation and generates tumour heterogeneity, thus leading to cancer metastasis, recurrence, and drug resistance. Although there has been substantial progress in CSC research into many solid tumour types, an understanding of the biology of CSCs in lung cancer remains elusive, mainly because of their heterogeneous origins and high plasticity. Here, we demonstrate that engineered lung cancer cells derived from normal human airway basal epithelial cells possessed CSC-like characteristics in terms of multilineage differentiation potential and strong tumour-initiating ability. Moreover, we established an in vitro 3D culture system that allowed the in vivo differentiation process of the CSC-like cells to be recapitulated. This engineered CSC model provides valuable opportunities for studying the biology of CSCs and for exploring and evaluating novel therapeutic approaches and targets in lung CSCs.

Integrated molecular analysis of adult T cell leukemia/lymphoma.

Adult T cell leukemia/lymphoma (ATL) is a peripheral T cell neoplasm of largely unknown genetic basis, associated with human T cell leukemia virus type-1 (HTLV-1) infection. Here we describe an integrated molecular study in which we performed whole-genome, exome, transcriptome and targeted resequencing, as well as array-based copy number and methylation analyses, in a total of 426 ATL cases. The identified alterations overlap significantly with the HTLV-1 Tax interactome and are highly enriched for T cell receptor-NF-κB signaling, T cell trafficking and other T cell-related pathways as well as immunosurveillance. Other notable features include a predominance of activating mutations (in PLCG1, PRKCB, CARD11, VAV1, IRF4, FYN, CCR4 and CCR7) and gene fusions (CTLA4-CD28 and ICOS-CD28). We also discovered frequent intragenic deletions involving IKZF2, CARD11 and TP73 and mutations in GATA3, HNRNPA2B1, GPR183, CSNK2A1, CSNK2B and CSNK1A1. Our findings not only provide unique insights into key molecules in T cell signaling but will also guide the development of new diagnostics and therapeutics in this intractable tumor.

A population of BJ fibroblasts escaped from Ras-induced senescence susceptible to transformation.

Oncogenic stimuli such as H-Ras induce oncogene-induced senescence (OIS) in fibroblasts to protect against transformation. Here we found that a population of the human diploid fibroblasts can escape from OIS induced by H-RasV12. We designated these OIS-escaped cells as OISEC (OIS-escaped cells). OISEC lost the expression of p16 which plays an important role for cell cycle arrest for induction of senescence, but OISEC preserved the p16 expression machinery and exhibited senescence by the treatment with hydrogen peroxide (H(2)O(2)) as stress-induced premature senescence (SIPS). OISEC did not possess anchorage-independent growth potential, and functional disruption of p53 and Rb by SV40 early region encoding large T and small t antigens, induced the aneuploidy phenotype and colony-forming potential of OISEC together with the exhibition of in vivo tumor formation. Finally, we also found that the distinctive feature of OISEC is expression of transcription factors, Oct3/4, SOX2, and Nanog which is closely related to stem-like cell features. This study highlights the presence of a cell population which escaped from OIS, and this OISEC may transform into malignant cancer cells by the additional hits of several genes in vivo.

Oncogene-mediated human lung epithelial cell transformation produces adenocarcinoma phenotypes in vivo.

It has been challenging to engineer lung adenocarcinoma models via oncogene-mediated transformation of primary cultured normal human cells. Although viral oncoprotein-mediated malignant transformation has been reported, xenografts derived from such transformed cells generally represent poorly differentiated cancers. Here, we demonstrate that the combined expression of multiple cellular factors induces malignant transformation in normal human lung epithelial cells. Although a combination of four genetic alterations, including hTERT overexpression, inactivation of the pRB and p53 pathways, and KRAS activation, is insufficient for normal human small airway epithelial cells to be fully transformed, expression of one additional oncogene induces malignant transformation. Notably, we have succeeded in reproducing human lung adenocarcinoma phenotypes in the flanks of nude mice by introducing an active form of PIK3CA, CYCLIN-D1, or a dominant-negative form of LKB1 in combination with the four genetic alterations above. Besides differentiated lung cancer, poorly differentiated cancer models can also be engineered by employing c-MYC as one of the genetic elements, indicating that histologic features and degree of differentiation of xenografts are controllable to some extent by changing the combination of genetic elements introduced. This is the first study reporting malignant transformation of normal lung epithelial cells in the absence of viral oncoproteins. We propose that our model system would be useful to identify the minimal and most crucial set of changes required for lung tumorigenesis, and that it would provide a broadly applicable approach for discovering attractive therapeutic targets.

Clinicopathologic application of lectin histochemistry: bisecting GlcNAc in glioblastoma.

Glycosylation is one of the most common posttranslational modifications and changes in oligosaccharide structures are associated with many human diseases including a number of cancers. Thus, discovering aberrant glycosylation patterns that serve as markers for brain tumor progression and metastasis represents an attractive strategy to improve clinicopathologic diagnosis and to provide aids to the development of novel therapies. To identify glioblastoma (GBM) cells expressing glycoproteins that contain high levels of the bisecting N-acetylglucosamine (GlcNAc) structures, lectin histochemistry was carried out using erythroagglutinating phytohemagglutinin. Although GBM frequently expressed the bisecting GlcNAc, the lectin reactivity varied among tumor regions within individual specimens. Since detailed histopathologic analysis revealed that oligosaccharides with bisecting GlcNAc structures were preferably expressed in tumor regions with low KI67 immunopositivity, immunodetection of the bisecting GlcNAc could be useful to indicate less proliferative regions in human GBM. Our study highlights the potential use of lectin histochemistry to develop new methods for diagnosis that would improve future antiglioma therapy.

Analysis of an alternative human CD133 promoter reveals the implication of Ras/ERK pathway in tumor stem-like hallmarks.

BACKGROUND: An increasing number of studies support the presence of stem-like cells in human malignancies. These cells are primarily responsible for tumor initiation and thus considered as a potential target to eradicate tumors. CD133 has been identified as an important cell surface marker to enrich the stem-like population in various human tumors. To reveal the molecular machinery underlying the stem-like features in tumor cells, we analyzed a promoter of CD133 gene using human colon carcinoma Caco-2 and synovial sarcoma Fuji cells, which endogenously express CD133 gene. RESULTS: A reporter analysis revealed that P5 promoter, located far upstream in a human CD133 gene locus, exhibits the highest activity among the five putative promoters (P1 to P5). Deletion and mutation analysis identified two ETS binding sites in the P5 region as being essential for its promoter activity. Electrophoretic mobility shift assays demonstrated the specific binding between nuclear factors and the ETS binding sequence. Overexpression of dominant-negative forms of Ets2 and Elk1 resulted in the significant decrease of P5 activity. Furthermore, treatment of Fuji cells with a specific MEK/ERK inhibitor, U0126, also markedly decreased CD133 expression, but there was no significant effect in Caco-2 cells, suggesting cell type-specific regulation of CD133 expression. Instead, the side population, another hallmark of TSLCs, was dramatically diminished in Caco-2 cells by U0126. Finally, Ras-mediated oncogenic transformation in normal human astrocytes conferred the stem-like capability to form neurosphere-like colonies with the increase of CD133 mRNA expression. CONCLUSIONS: In conclusion, the Ras/ERK pathway at least in part contributes to the maintenance and the acquisition of stem-like hallmarks, although the extent of its contribution is varied in a cell type-specific manner. These findings could help our comprehensive understanding of tumor stemness, and also improve the development of eradicative therapies against human malignancies.

Overexpression of caveolin-1 in adult T-cell leukemia.

Caveolin-1 is implicated in the regulation of signal pathways. Adult T-cell leukemia (ATL) is a T-cell malignancy causatively associated with human T-cell leukemia virus type 1 (HTLV-1). To determine the role of caveolin-1 in leukemogenesis, we examined caveolin-1 expression levels in HTLV-1-infected T-cell lines and ATL cells. These cells expressed high levels of caveolin-1 compared with uninfected T-cell lines and normal peripheral blood mononuclear cells (PBMCs). Caveolin-1-positive ATL cells were detected in ATL lymph nodes and skin lesions, and caveolin-1 was also detected in the plasma of patients with ATL. Infection of a human T-cell line, an epithelial cell line, and normal PBMCs with HTLV-1 induced caveolin-1 expression. The viral protein Tax transcriptionally activated caveolin-1 gene through nuclear factor-kappaB and cAMP response element binding protein signal pathways. HTLV-1-infected T-cell lines, and ATL cells are known to be resistant to transforming growth factor beta (TGF-beta)-induced growth inhibition. Caveolin-1 was colocalized with TGF-beta type I receptor in HTLV-1-infected T-cell lines and suppressed TGF-beta signaling. Caveolin-1 knockdown in an HTLV-1-infected T-cell line exhibited susceptibility to TGF-beta. Thus, we describe a new function for Tax, repression of TGF-beta signaling through caveolin-1 expression, which may play a critical role in ATL leukemogenesis.

Promoter hypomethylation regulates CD133 expression in human gliomas.

Brain tumor-initiating cells (BTICs) have been enriched using antibodies against the cell surface protein CD133; however, the biological relevance and the regulatory mechanism of CD133 expression in human gliomas are not yet understood. In this study, we initially demonstrated that CD133 was overexpressed in high-grade human glioblastomas where CD133-positive cells were focally observed as a micro-cluster. In addition, CD133 transcripts with exon 1A, 1B, or 1C were predominantly expressed in glioblastomas. To elucidate the mechanism regulating this aberrant expression of CD133, three proximal promoters (P1, P2, and P3) containing a CpG island were isolated. In U251MG and T98G glioblastoma cells, the P1 region flanking exon 1A exhibited the highest activity among the three promoters, and this activity was significantly inactivated by in vitro methylation. After treatment with the demethylating agent 5-azacytidine and/or the histone deacetylase inhibitor valproic acid, the expression level of CD133 mRNA was significantly restored in glioma cells. Importantly, hypomethylation of CpG sites within the P1, P2, and P3 regions was observed by bisulfite sequencing in human glioblastoma tissues with abundant CD133 mRNA. Taken together, our results indicate that DNA hypomethylation is an important determinant of CD133 expression in glioblastomas, and this epigenetic event may be associated with the development of BTICs expressing CD133.

Careful exclusion of non-neoplastic brain components is required for an appropriate evaluation of O6-methylguanine-DNA methyltransferase status in glioma: relationship between immunohistochemistry and methylation analysis.

Evaluation of O6-methylguanine-DNA methyltransferase (MGMT) expression is important for antiglioma therapy as many clinical trials have demonstrated that promoter hypermethylation and low level expression of MGMT are associated with an enhanced response to alkylating agents. However, here we report that the current strategies used to evaluate MGMT status in gliomas are unreliable. We observed discordance in the MGMT expression status when immunohistochemical evaluation and polymerase chain reaction-based methylation assessments were used: 73% of gliomas with methylated MGMT promoter had substantial numbers of MGMT-immunopositive tumor cells. Furthermore, when MGMT expression was tested in tumor homogenates using reverse transcription-polymerase chain reaction, 43% of tumors were found positive, in comparison to only 24%, when histologic samples were assayed immunohistochemically. To explain these inconsistencies we undertook a detailed immunohistochemical evaluation of tumor samples and found that some gliomas demonstrated remarkably high expression of MGMT in the entire tumor whereas others contained only a small immunopositive area. Additionally, we found that gliomas contained various types of non-neoplastic cells expressing MGMT, including lymphocytes, vascular endothelial cells, and macrophages/microglias, which contribute to overall MGMT expression detected in tumor homogenates, and thus result in overestimation of tumor MGMT expression. Therefore, to correctly establish MGMT expression in the tumor, which could be informative of glioma sensitivity to alkylating agents, exclusion of non-neoplastic brain components from analysis is required.

Establishment of a luciferase assay-based screening system: fumitremorgin C selectively inhibits cellular proliferation of immortalized astrocytes expressing an active form of AKT.

The AKT pathway is frequently activated in glioblastoma, and as such, inhibitors of this pathway could prove very useful as anti-glioblastoma therapies. Here we established immortalized astrocytes expressing Renilla luciferase as well as those expressing both an active form of AKT and firefly luciferase. Since both luciferase activities represent the numbers of corresponding cell lines, novel inhibitors of the AKT pathway can be identified by treating co-cultures containing the two types of luciferase-expressing cells with individual compounds. Indeed, such a screening system succeeded in identifying fumitremorgin C as an efficient inhibitor of the AKT pathway, which was further confirmed by the ability of fumitremorgin C to selectively inhibit the growth of immortalized astrocytes expressing an active form of AKT. The present study proposes a broadly applicable approach for identifying therapeutic agents that target the pathways and/or molecules responsible for cancer development.

O6-methylguanine-DNA methyltransferase is downregulated in transformed astrocyte cells: implications for anti-glioma therapies.

BACKGROUND: A novel alkylating agent, temozolomide, has proven efficacious in the treatment of malignant gliomas. However, expression of O6-methylguanine-DNA methyltransferase (MGMT) renders glioma cells resistant to the treatment, indicating that identification of mechanisms underlying the gene regulation of MGMT is highly required. Although glioma-derived cell lines have been widely employed to understand such mechanisms, those models harbor numerous unidentified genetic lesions specific for individual cell lines, which complicates the study of specific molecules and pathways. RESULTS: We established glioma models by transforming normal human astrocyte cells via retroviral-mediated gene transfer of defined genetic elements and found that MGMT was downregulated in the transformed cells. Interestingly, inhibitors of DNA methylation and histone deacetylation failed to increase MGMT protein levels in the transformed astrocyte cells as well as cultured glioblastoma cell lines, whereas the treatment partially restored mRNA levels. These observations suggest that downregulation of MGMT may depend largely on cellular factors other than promoter-hypermethylation of MGMT genes, which is being used in the clinic to nominate patients for temozolomide treatment. Furthermore, we discovered that Valproic acid, one of histone deacetylase inhibitors, suppressed growth of the transformed astrocyte cells without increasing MGMT protein, suggesting that such epigenetic compounds may be used to some types of gliomas in combination with alkylating agents. CONCLUSION: Normal human astrocyte cells allow us to generate experimental models of human gliomas by direct manipulation with defined genetic elements, in contrast to tumor-derived cell lines which harbor numerous unknown genetic abnormalities. Thus, we propose that the study using the transformed astrocyte cells would be useful for identifying the mechanisms underlying MGMT regulation in tumor and for the development of rational drug combination in glioma therapies.

Medulloblastomas derived from Cxcr6 mutant mice respond to treatment with a smoothened inhibitor.

The sonic hedgehog (Shh) pathway is activated in approximately 30% of human medulloblastoma resulting in increased expression of downstream target genes. In about half of these cases, this has been shown to be a consequence of mutations in regulatory genes within the pathway, including Ptc1, Smo, and Sufu. However, for some tumors, no mutations have been detected in known pathway genes. This suggests that either mutations in other genes promote tumorigenesis or that epigenetic alterations increase pathway activity in these tumors. Here, we report that 3% to 4% of mice lacking either one or both functional copies of Cxcr6 develop medulloblastoma. Although CXCR6 is not known to be involved in Shh signaling, tumors derived from Cxcr6 mutant mice expressed Shh pathway target genes including Gli1, Gli2, Ptc2, and Sfrp1, indicating elevated pathway activity. Interestingly, the level of Ptc1 expression was decreased in tumor cells although two normal copies of Ptc1 were retained. This implies that reduced CXCR6 function leads to suppression of Ptc1 thereby increasing Smoothened function and promoting tumorigenesis. We used a direct transplant model to test the sensitivity of medulloblastoma arising in Cxcr6 mutant mice to a small-molecule inhibitor of Smoothened (HhAntag). We found that transplanted tumors were dramatically inhibited in mice treated for only 4 days with HhAntag. These findings suggest that HhAntag may be effective against tumors lacking mutations in known Shh pathway genes.

Shh pathway activity is down-regulated in cultured medulloblastoma cells: implications for preclinical studies.

Gene expression profiling indicates that the Sonic Hedgehog (Shh) pathway is active in approximately 30% of human medulloblastomas, suggesting that it could provide a useful therapeutic target. Previously, we showed that spontaneous medulloblastomas in Ptc1(+/-)p53-/- mice could be eradicated by treatment with a small-molecule inhibitor (HhAntag) of Smoothened (Smo). Here, we compared the responses of mouse medulloblastoma cells propagated in flank allografts, either directly or after culture in vitro, to HhAntag. We found that Shh pathway activity was suppressed in medulloblastoma cells cultured in vitro and it was not restored when these cells were transplanted into the flank of nude mice. The growth of these transplanted tumor cells was not inhibited by treatment of mice with doses of HhAntag that completely suppressed Smo activity. Interestingly, tumor cells transplanted directly into the flank maintained Smo activity and were sensitive to treatment with HhAntag. These findings indicate that propagation of tumor cells in culture inhibits Smo activity in a way that cannot be reversed by transplantation in vivo, and they raise concerns about the use of cultured tumor cells to test the efficacy of Shh pathway inhibitors as anticancer therapies.

FRA1 is a determinant for the difference in RAS-induced transformation between human and rat fibroblasts.

Human diploid fibroblasts (HDF) immortalized by hTERT and simian virus 40 (SV40) early region (ER) exhibit a limited degree of transformation upon the expression of activated H-RAS (H-RAS V12) compared with rat embryonic fibroblasts (REF) immortalized by SV40 ER. Here, we identified FRA1 as a determinant for this difference in RAS-induced transformation. FRA1 was not induced by H-RAS V12 in the immortalized HDF, in contrast to its marked accumulation in the immortalized REF. Ectopic expression of FRA1 significantly enhanced anchorage-independent growth of various HDF expressing hTERT, SV40 ER, and H-RAS V12. More importantly, FRA1 could induce anchorage-independent growth as well as nude mice tumor formation of the immortalized HDF in the absence of H-RAS V12. The results of an in vitro kinase assay clearly showed that the RAS-induced extracellular signal-regulated kinase (ERK) activation, which is responsible for FRA1 induction, was markedly attenuated in the HDF compared with that in the REF, despite no obvious differences in the phosphorylation status of ERK between the species. Our results strongly suggest that HDF negatively regulate the mitogen-activated protein kinase kinase (MEK)/ERK pathway more efficiently than REF, and consequently express less malignant phenotypes in response to H-RAS V12.

Suppression of the Shh pathway using a small molecule inhibitor eliminates medulloblastoma in Ptc1(+/-)p53(-/-) mice.

Medulloblastoma is the most common malignant pediatric brain tumor. Current treatment is associated with major long-term side effects; therefore, new nontoxic therapies, targeting specific molecular defects in this cancer, need to be developed. We use a mouse model of medulloblastoma to show that inhibition of the Sonic Hedgehog (Shh) pathway provides a novel therapy for medulloblastoma. A small molecule inhibitor of the Shh pathway, HhAntag, blocked the function of Smoothened in mice with medulloblastoma. This resulted in suppression of several genes highly expressed in medulloblastoma, inhibition of cell proliferation, increase in cell death and, at the highest dose, complete eradication of tumors. Long-term treatment with HhAntag prolonged medulloblastoma-free survival. These findings support the development of Shh antagonists for the treatment of medulloblastoma.

Refractory nature of normal human diploid fibroblasts with respect to oncogene-mediated transformation.

Human cells are known to be more refractory than rodent cells against oncogenic transformation in vitro. To date, the molecular mechanisms underlying such resistance remain largely unknown. The combination of simian virus 40 early region and H-Ras V12 has been effective for transformation of rat embryo fibroblasts, but not for human cells. However, the additional ectopic expression of the telomerase catalytic subunit (hTERT) was reported to be capable of causing transformation of normal human cells. In this study, however, we demonstrate that the combined expression of the above-mentioned three genetic elements is not always sufficient to transform normal human diploid fibroblasts (HDF). Although the expression and function of these introduced genetic elements were essentially the same, among four HDF, TIG-1 and TIG-3 were resistant to transformation. The other two (BJ and IMR-90) showed transformed phenotypes, but they were much restricted compared with rat embryo fibroblasts in expressing simian virus 40 early region and H-Ras V12. In correlation with these phenotypes, TIG-1 and TIG-3 remained diploid after the introduction of these genetic elements, whereas BJ and IMR-90 became highly aneuploid. These results strongly suggest that the lack of telomerase is not the sole reason for the refractory nature of HDF against transformation and that normal human cells have still undefined intrinsic mechanisms rendering them resistant to oncogenic transformation.