Immortalization of normal human mammary epithelial cells in two steps by direct targeting of senescence barriers does not require gross genomic alterations

Telomerase reactivation and immortalization are critical for human carcinoma progression. However, little is known about the mechanisms controlling this crucial step, due in part to the paucity of experimentally tractable model systems that can examine human epithelial cell immortalization as it might occur in vivo. We achieved efficient non-clonal immortalization of normal human mammary epithelial cells (HMEC) by directly targeting the 2 main senescence barriers encountered by cultured HMEC. The stress-associated stasis barrier was bypassed using shRNA to p16^INK4; replicative senescence due to critically shortened telomeres was bypassed in post-stasis HMEC by c-MYC transduction. Thus, 2 pathologically relevant oncogenic agents are sufficient to immortally transform normal HMEC. The resultant non-clonal immortalized lines exhibited normal karyotypes. Most human carcinomas contain genomically unstable cells, with widespread instability first observed in vivo in pre-malignant stages; in vitro, instability is seen as finite cells with critically shortened telomeres approach replicative senescence. Our results support our hypotheses that: (1) telomere-dysfunction induced genomic instability in pre-malignant finite cells may generate the errors required for telomerase reactivation and immortalization, as well as many additional “passenger” errors carried forward into resulting carcinomas; (2) genomic instability during cancer progression is needed to generate errors that overcome tumor suppressive barriers, but not required per se; bypassing the senescence barriers by direct targeting eliminated a need for genomic errors to generate immortalization. Achieving efficient HMEC immortalization, in the absence of “passenger” genomic errors, should facilitate examination of telomerase regulation during human carcinoma progression, and exploration of agents that could prevent immortalization.     

Immortalization and transformation of human cells

The disruption of homeostatic mechanisms that regulate normal cell growth and proliferation is a hallmark of cancer. Experimentally, many of the same genetic changes that lead to abnormal cell proliferation conspire to confer replicative immortality upon cells in culture. Correspondingly, several lines of evidence implicate cellular immortalization as a prerequisite for cell transformation. Recently much progress has been made in elucidating the cellular machinery that regulates cell lifespan. This review summarizes these recent advances in our understanding of these molecular mechanisms that contribute to human cell immortalization and transformation.     

The induction of growth arrest in fibroblasts by SV40 T antigen

DNA tumor viruses such as SV40, Ras and papillomaviruses are the most commonly used agents in immortalization of non-hematopoietic cells, but the results are quite different. Some of them even lead instead to a senescence-like state. To verify the potential of SV40 T antigen-mediated immortalization or properties and functions of it to regulate cell growth, human dermal fibroblasts were cultured and then transfected with eukaryotic expressing plasmid psv3-neo which containing SV40 T DNA. We found that expression of oncogenic SV40 T in human dermal fibroblasts resulted in growth, arrest, earlier than the occurrence of control cell senescence, although telomerase was positive and cells grew faster than control ones in early stage following transfection. These observations suggest that SV40 T antigen can activate growth arrest in human dermal fibroblasts under normal growth condition instead of always prolonging the lifespan of fibroblasts. Moreover, high rate of cell division in early stage after transfection may be associated with the expression of telomerase activity.     

Differential gene expression in SV40-mediated immortalization of human fibroblasts

Normal human diploid fibroblasts (HF) have a limited life span, undergo senescence, and rarely, if ever, spontaneously immortalize in culture. Introduction of the gene for T antigen encoded by the DNA virus SV40 extends the life span of HF and increases the frequency of immortalization; however, immortalization requires both T-dependent and T-independent functions. We previously generated independent SV40-transformed non-immortal (pre-immortal) HF cell lines from which we then obtained immortal sublines as part of a multifaceted approach to identify functions responsible for immortalization. In this study we undertook a search for cellular mRNAs which are differentially expressed upon immortalization. A λcDNA library was prepared from a pre-immortal SV40-transformed HF (HF-C). We screened the library with a subtracted probe enriched for sequences present in HF-C and reduced in immortal AR5 cells. A more limited screen was also employed for sequences overexpressed in AR5 using a different strategy. Alterations in the level of mRNAs in AR5 encoding functions relevant to signal transduction pathways were identified; however, most cDNAs encoded novel sequences. In an effort to clarify which of the altered mRNAs are most relevant to immortalization, we performed Northern analysis with RNA prepared from three paired sets of independent pre-immortal and immortal (4 cell lines) SV40-transformants using eight cloned cDNAs which show reduced expression in AR5. Three of these were reduced in additional immortal cell lines as well; one, J4-4 (unknown function) is reduced in all the immortal cell lines tested; a second, J4-3 (possible PP2C type phosphatase) is reduced in 2 of the 3 matched sets; and a third, J2-2 (unknown function) is redu ced in 2 unrelated immortal cell lines. Although the roles of these genes are as yet unclear, their further analysis should extend our understanding of the molecular bases for immortalization. In particular, the patterns of expression of J4-4 and J4-3 strongly suggest that they are involved in the process of immortalization and/or can serve as target genes for assessing regulators of gene expression in this process. J. Cell. Physiol. 171:325–335, 1997. © 1997 Wiley-Liss, Inc.     

人B细胞永生化研究进展

人源单克隆抗体具有免疫原性低、半衰期长等优势,成为了体内应用中不可或缺的生物制剂.人类抗体库为人源单克隆抗体的制备提供了丰富的来源,人B细胞永生化是获得人类抗体库的潜在有效方法,可应用于人源单克隆抗体的制备.由于各平台均有亟待解决的问题,基于人B细胞永生化的抗体制备尚局限在实验室研究阶段,且目前尚缺乏一篇系统综述以明确...     

Characterization of envelope glycoprotein mutants for human T-cell leukemia virus type 1 infectivity and immortalization

The human T-cell leukemia virus type 1 (HTLV-1) envelope protein is required for virus spread. This study further characterizes the role of the envelope protein in HTLV-1 immortalization. Viruses with single amino acid substitutions within the SU protein at residue 75, 81, 95, 101, 105, or 195 or with a C-terminal cytoplasmic domain truncation (CT), as well as an envelope-null (EN) virus, were generated within an infectious molecular clone, ACH. Transfection of 293T cells resulted in the release of similar amounts of virus particles from all of the mutants as determined by p19 enzyme-linked immunosorbent assay and immunoblot analysis of Gag in cell lysates and supernatants. The virus particles from all mutants except ACH-101, ACH-CT, and ACH-EN were infectious for B5 macaque cells in cell-free and cell-to-cell transmission assays and were capable of immortalizing transfected CD4(+) lymphocytes. These results indicate that HTLV-1 spread is required for immortalization.     

Telomerase induces immortalization of human esophageal keratinocytes without p16INK4a inactivation

Normal human somatic cells have a finite life span and undergo replicative senescence after a limited number of cell divisions. Erosion of telomeric DNA has emerged as a key factor in senescence, which is antagonized during cell immortalization and transformation. To clarify the involvement of telomerase in the immortalization of keratinocytes, catalytic subunit of telomerase (hTERT) expression was restored in normal human esophageal epithelial cells (EPC2). EPC2-hTERT cells overcame senescence and were immortalized without p16INK4a genetic or epigenetic alterations. p16INK4a was expressed at moderate levels and remained functional as evidenced by induction with UV treatment and binding to cyclin-dependent kinase 4 and 6. There were no mutations in the p53 gene, and p53 was functionally intact. Importantly, senescence could be activated in the immortalized EPC2-hTERT cells by overexpression of oncogenic H-ras or p16INK4a. Furthermore, the EPC2-hTERT cells yielded basal cell hyperplasia in an innovative organotypic culture system in contrast to a normal epithelium from parental cells. These comprehensive results indicate that the expression of telomerase induces immortalization of normal human esophageal keratinocytes without inactivation of p16INK4a/pRb pathway or abrogation of the p53 pathway.     

Telomeres, telomerase and malignant transformation

Human cancer arises in a stepwise process by the accumulation of genetic alterations in oncogenes, tumor suppressor genes and other genes involved in the regulation of cell growth and proliferation. Many genes, important for the pathogenesis of various cancers and the pathways through which they act, have been characterized over the past decades. Nevertheless, recent successes in experimental models of immortalization and malignant transformation of human cells indicate that the disruption of a limited number of cellular pathways is sufficient to induce a cancerous phenotype in a wide variety of normal cells. In this context, immortalization is an essential prerequisite for the formation of a tumor cell. Besides classical cancer related pathways as the pRB and p53 tumor suppressor pathway or the ras signaling pathway, the maintenance of telomeres plays an essential role in both of these processes. Alterations in telomere biology both suppress and facilitate malignant transformation by regulating genomic stability and cellular life span. This review will summarize recent advances in the understanding of the molecular mechanisms of malignant transformation in human cells and the role of telomere maintenance in these processes. This ultimately leads to the development of cellular models of human cancer that phenocopy the corresponding disease. Furthermore, in the future these models could provide an ideal basis for the testing of novel chemopreventive or therapeutic approaches in the treatment of different types of human cancer.     

Finite life span of hybrids formed by fusion of different simian virus 40-immortalized human cell lines.

Simian virus 40 (SV40) genes are able to induce immortalization of normal human cells after a culture crisis during which unknown cellular genetic changes presumably occur. To determine whether these genetic changes are always identical, we performed somatic cell hybridization analysis of an SV40-immortalized human bronchial epithelial cell line, BET-1A. Fusion of BET-1A with an SV40-immortalized fibroblast cell line resulted in hybrids that senesced, indicating that these cell lines are in different complementation groups for immortalization.     

MicroRNA-296 is enriched in cancer cells and downregulates p21WAF1 mRNA expression via interaction with its 3' untranslated region

MicroRNAs (miRNAs) are a class of noncoding small RNAs that act as negative regulators of gene expression. To identify miRNAs that may regulate human cell immortalization and carcinogenesis, we performed comparative miRNA array profiling of human normal and SV40-T antigen immortalized cells. We found that miR-296 was upregulated in immortalized cells that also had activation of telomerase. By an independent experiment on genomic analysis of cancer cells we found that chromosome region (20q13.32), where miR-296 is located, was amplified in 28/36 cell lines, and most of these showed enriched miR-296 expression. Overexpression of miR-296 in human cancer cells, with and without telomerase activity, had no effect on their telomerase function. Instead, it suppressed p53 function that is frequently downregulated during human cell immortalization and carcinogenesis. By monitoring the activity of a luciferase reporter connected to p53 and p21(WAF1) (p21) untranslated regions (UTRs), we demonstrate that miR-296 interacts with the p21-3'UTR, and the Hu binding site of p21-3'UTR was identified as a potential miR-296 target site. We demonstrate for the first time that miR-296 is frequently upregulated during immortalization of human cells and contributes to carcinogenesis by downregulation of p53-p21(WAF1) pathway.     

Telomere elongation in immortal human cells without detectable telomerase activity.

Immortalization of human cells is often associated with reactivation of telomerase, a ribonucleoprotein enzyme that adds TTAGGG repeats onto telomeres and compensates for their shortening. We examined whether telomerase activation is necessary for immortalization. All normal human fibroblasts tested were negative for telomerase activity. Thirteen out of 13 DNA tumor virus-transformed cell cultures were also negative in the pre-crisis (i.e. non-immortalized) stage. Of 35 immortalized cell lines, 20 had telomerase activity as expected, but 15 had no detectable telomerase. The 15 telomerase-negative immortalized cell lines all had very long and heterogeneous telomeres of up to 50 kb. Hybrids between telomerase-negative and telomerase-positive cells senesced. Two senescent hybrids demonstrated telomerase activity, indicating that activation of telomerase is not sufficient for immortalization. Some hybrid clones subsequently recommenced proliferation and became immortalized either with or without telomerase activity. Those without telomerase activity also had very long and heterogeneous telomeres. Taken together, these data suggest that the presence of lengthened or stabilized telomeres is necessary for immortalization, and that this may be achieved either by the reactivation of telomerase or by a novel and as yet unidentified mechanism.     

Contribution of human data to the analysis of human carcinogenesis

Human data strongly suggest that small doses or low concentrations of genotoxic agents cause only a relatively small number of human cancers. They emphasize the role of promotion, in particular that associated with cell proliferation. There is therefore a qualitative difference between high doses of genotoxic agents which provoke cell death and a compensatory increase in cell division, and low doses which do not. During the promotion phase, human data demonstrate the importance of induced genetic instability and defects in apoptosis as well as that of cell immortalization which play a main role for the accumulation in a cell genome of several specific lesions. Carcinogenesis is a complex process in which initial mutations do not appear to be a limiting or crucial step. This view is supported by the paramount influence of age on the induction by radiation of thyroid and breast cancer. It is also compatible with practical thresholds observed in subjects whose bones or liver were exposed to alpha-emitters, as well as with the curvilinearity in the leukemia incidence dose-response in the Japanese atomic bomb survivors. The linear no threshold model assumes that: 1) the probability of DNA lesion repair is constant whatever the dose and, hence, the number of lesions provoked in the same cell and the surrounding cells; 2) the probability for a damaged cell to evolve toward an invasive cancer is not influenced by the possible promotional effect of further irradiation or induced tissue proliferation, nor the control exerted by surrounding cells. These assumptions deserve a critical analysis.     

肝细胞永生化策略研究进展

正常体细胞在细胞分裂过程中由于无法维持端粒的长度和染色体的稳定而衰老、死亡。建立基因稳定、无致瘤性的永生化细胞是永生化研究的目的所在。利用Cre-loxP系统构建的可恢复性永生化细胞可用于以细胞为基础的临床治疗。分化良好并具有肝脏正常合成代谢功能的永生化肝细胞在生物医学研究,尤其是在肝细胞移植、生物人工肝支持治疗以及药理学和毒理学研究方面具有广泛的应用前景。     

Immortalization of mutant p53-transfected human fibroblasts by treatment with either 4-nitroquinoline 1-oxide or x-rays

Summary The study of in vitro cell transformation is valuable for understanding the multistep carcinogenesis of human cells. The difficulty in inducing neoplastic transformation of human cells by treatment with chemical or physical agents alone is due to the difficulty in immortalizing normal human cells. Thus, the immortalization step is critical for in vitro neoplastic transformation of human cells. We transfected a mutant p53 gene (mp53: codon 273^Arg-His) into normal human fibroblasts and obtained two G418-resistant mp53-containing clones. These clones showed an extended life span but ultimately senesced. However, when they were treated with either 4-nitroquinoline 1-oxide or X rays, they were immortalized. The immortalized cells showed both numerical and structural chromosome abnormalities, but they were not tumorigenic. The expression of mutant but not wild type p53 was detected in the immortalized cells by RT-PCR. Expression of p21, which is located downstream of p53, was remarkably reduced in the immortalized cells, resulting in increased cdk2 and cdc2 kinase activity. However, there was no significant difference between the normal and immortalized human cells in expression of another tumor suppressor gene, p16. These findings indicate that the p53-p21 cascade may play an important role in the immortalization of human cells.     

A method for transforming lymphocytes from very small blood volumes suitable for paediatric samples

Summary Permanent lymphoblastoid cell lines are important in the molecular analysis and characterization of human genetic disorders, when immortalized cells must be banked for future diagnostic or research purposes. However, routine methods for transformation using Epstein-Barr virus (EBV) require blood volumes that may be difficult to collect from clinically compromised neonates and small children. Here we report a modified transformation procedure utilizing blood samples of small volume (less than 1.0ml), which we have found to be particularly useful for the immortalization of lymphocytes destined for future molecular genetic studies.