Immortalization in a normal foreskin fibroblast culture following transduction of cyclin A2 or cdk1 genes in retroviral vectors

Human diploid fibroblasts (HDF) rarely, if ever, undergo spontaneous transformation to an immortalized cell type. Here we report the immortalization of an HDF cell line following transduction with cyclin A2 or cdk1 human genes via retroviral vectors. Fluorescence in situ hybridization (FISH) studies using the retroviral vector as a probe indicate that these cell lines are monoclonal. No telomerase activity could be detected in these cell lines, and the telomere length in the immortalized cells was observed to be 10-20 kb longer than that in low-passage cells from the parental fibroblast line. Cytogenetic studies revealed that the immortal lines share common chromosomal aberrations. FISH studies with a probe for p53 revealed loss of one copy of this gene which was associated with reduced steady-state levels of both p53 and p53-regulated p21(WAF1/Sdi1/CIP1) messages in both quiescent and proliferating immortalized cultures relative to the parental cells. Additional FISH studies with probes for p16(INK4a) and Rb, carried out after the immortalized cells proliferated in excess of 100 population doublings, also revealed loss of one copy of these genes in both cell lines. These cell lines, together with the well-characterized parental cells, could provide useful research material for the study of the mechanisms of immortalization and of regulation of proliferative senescence in HDF.     

MiR-24 Tumor Suppressor Activity Is Regulated Independent of p53 and through a Target Site Polymorphism

MicroRNAs (miRNAs) are predicted to regulate approximately 30% of all human genes; however, only a few miRNAs have been assigned their targets and specific functions. Here we demonstrate that miR-24, a ubiquitously expressed miRNA, has an anti-proliferative effect independent of p53 function. Cell lines with differential p53 status were used as a model to study the effects of miR-24 on cell proliferation, cell cycle control, gene regulation and cellular transformation. Overexpression of miR-24 in six different cell lines, independent of p53 function, inhibited cell proliferation and resulted in G2/S cell cycle arrest. MiR-24 over expression in cells with wt-p53 upregulated TP53 and p21 protein; however, in p53-null cells miR-24 still induced cell cycle arrest without the involvement of p21. We show that miR-24 regulates p53-independent cellular proliferation by regulating an S-phase enzyme, dihydrofolate reductase (DHFR) a target of the chemotherapeutic drug methotrexate (MTX). Of interest, we found that a miR-24 target site polymorphism in DHFR 3′ UTR that results in loss of miR-24-function and high DHFR levels in the cell imparts a growth advantage to immortalized cells and induces neoplastic transformation. Of clinical significance, we found that miR-24 is deregulated in human colorectal cancer tumors and a subset of tumors has reduced levels of miR-24. A novel function for miR-24 as a p53-independent cell cycle inhibitory miRNA is proposed.     

Telomerase-immortalized human fibroblasts retain UV-induced mutagenesis and p53-mediated DNA damage responses

Immortalized cells frequently have disruptions of p53 activity and lack p53-dependent nucleotide excision repair (NER). We hypothesized that telomerase immortalization would not alter p53-mediated ultraviolet light (UV)-induced DNA damage responses. DNA repair proficient primary diploid human fibroblasts (GM00024) were immortalized by transduction with a telomerase expressing retrovirus. Empty retrovirus transduced cells senesced after a few doublings. Telomerase transduced GM00024 cells (tGM24) were cultured continuously for 6 months (>60 doublings). Colony forming ability after UV irradiation was dose-dependent between 0 and 20J/m2 UVC (LD50=5.6J/m2). p53 accumulation was UV dose- and time-dependent as was induction of p48(XPE/DDB2), p21(CIP1/WAF1), and phosphorylation on p53-S15. UV dose-dependent apoptosis was measured by nuclear condensation. UV exposure induced UV-damaged DNA binding as monitored by electrophoretic mobility shift assays using UV irradiated radiolabeled DNA probe was inhibited by p53-specific siRNA transfection. p53-Specific siRNA transfection also prevented UV induction of p48 and improved UV survival measured by colony forming ability. Strand-specific NER of cyclobutane pyrimidine dimers (CPD) within DHFR was identical in tGM24 and GM00024 cells. CPD removal from the transcribed strand was nearly complete in 6h and from the non-transcribed strand was 73% complete in 24h. UV-induced HPRT mutagenesis in tGM24 was indistinguishable from primary human fibroblasts. These wide-ranging findings indicate that the UV-induced DNA damage response remains intact in telomerase-immortalized cells. Furthermore, telomerase immortalization provides permanent cell lines for testing the immediate impact on NER and mutagenesis of selective genetic manipulation without propagation to establish mutant lines.     

P53-dependent miRNAs mediate nitric oxide-induced apoptosis in colonic carcinogenesis

Both miRNAs and nitric oxide (NO) play important roles in colonic inflammation and tumorigenesis. Resistance of colonic epithelial cells to apoptosis may contribute to tumor development. We hypothesized that some miRNAs could increase the resistance of colonic cancer cells to nitric oxide-induced apoptotic cell death. Here we show that NO induced apoptosis and stimulated expression of some miRNAs. Loss of p53 not only blocked NO-induced apoptosis but also dramatically inhibited the expression of NO-related miRNAs, such as miR-34, miR-203, and miR-1301. In addition, blockage of p53-dependent miRNAs significantly reduced NO-induced apoptosis. Furthermore, forced expression of these miRNAs rendered HT-29 cells, which are resistant to apoptosis with mutant p53, more sensitive to NO-induced apoptotic cell death. Most interestingly, in a colitis-associated colon cancer mouse model, the level of miRNAs dropped significantly, accompanied by downregulation of p21, which is a key target gene of p53. In human colorectal cancer samples, the expression of miR-34 significantly correlated with the level of inducible nitric oxide synthase (iNOS). We contend that increased NO production may select cells with low levels of p53-dependent miRNAs which contributes to human colonic carcinogenesis and tumor progression.     

EB病毒感染人胎鼻咽上皮细胞逃避老化期过程中抑制p16INK4a表达

细胞周期调控紊乱是细胞永生化进程中一个重要的分子事件,本文利用Western blotting和S-P法分别检测p16~(INK4a)、p53、p21~(WAF1/CIP1)和E2F1的蛋白表达,试图从细胞周期调控的角度,探讨EB病毒诱导人胎鼻咽上皮细胞逃避老化期的分子机制。结果表明,EB病毒通过抑制p16~(INK4a)表达而阻断p16~(INK4a)/Rb途径,上调转录因子E2F1,而对p53、p21~(WAF1/CIP1)表达无明显的影响。结果初步揭示,EB病毒介导的p16~(INK4a)/Rb/E2F1细胞周期调控紊乱参与了人胎鼻咽上皮细胞逃避老化期过程,为进一步探讨鼻咽癌发病机制提供了科学依据。     

EB病毒感染人胎鼻咽上皮细胞逃避老化期过程中抑制p16^INK4a表达

细胞周期调控紊乱是细胞永生化进程中一个重要的分子事件,本文利用Western blotting和S-P法分别检测p16^INK4a、p53、p21^WAF1／CIP1和E2F1的蛋白表达,试图从细胞周期调控的角度,探讨EB病毒诱导人胎鼻咽上皮细胞逃避老化期的分子机制。结果表明,EB病毒通过抑制p16^INK4a表达而阻断p16^INK4a／Rb途径,上调转录因子E2F1,而对p53、p21^WAF1／CIP表达无明显的影响。结果初步揭示,EB病毒介导的p16^INK4a／Rb／E2F1细胞周期调控紊乱参与了人胎鼻咽上皮细胞逃避老化期过程,为进一步探讨鼻咽癌发病机制提供了科学依据。     

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.     

Maintenance of telomeres in SV40-transformed pre-immortal and immortal human fibroblasts

Shortening of telomeres has been hypothesized to contribute to cellular senescence and may play a role in carcinogenesis of human cells. Furthermore, activation of telomerase has frequently been demonstrated in tumor-derived and in vitro immortalized cells. In this study, we have assessed these phenomena during the life span of simian virus 40 (SV40)-transformed preimmortal and immortal human fibroblasts. We observed progressive reduction in telomere length in preimmortal transformed cells with extended proliferative capacity, with the most dramatic shortening at late passage. Telomere lengths became stabilized (or increased) in immortal fibroblasts accompanied, in one case, by the activation of telomerase. However, an independent immortal cell line that displayed stable telomeres did not have detectable telomerase activity. Furthermore, we found significant telomerase activity in two preimmortal derivatives. Our results provide further evidence for maintenance of telomeres in immortalized human fibroblasts, but they suggest a lack of causal relationship between telomerase activation and immortalization. © 1996 Wiley-Liss, Inc.     

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.     

Upregulation of miR-760 and miR-186 Is Associated with Replicative Senescence in Human Lung Fibroblast Cells

We have previously shown that microRNAs (miRNAs) miR-760, miR-186, miR-337-3p, and miR-216b stimulate premature senescence through protein kinase CK2 (CK2) down-regulation in human colon cancer cells. Here, we examined whether these four miRNAs are involved in the replicative senescence of human lung fibroblast IMR-90 cells. miR-760 and miR-186 were significantly upregulated in replicatively senescent IMR-90 cells, and their joint action with both miR-337-3p and miR-216b was necessary for efficient downregulation of the α subunit of CK2 (CK2α) in IMR-90 cells. A mutation in any of the four miRNA-binding sequences within the CK2α 3′-untranslated region (UTR) indicated that all four miRNAs should simultaneously bind to the target sites for CK2α downregulation. The four miRNAs increased senescence-associated β-galactosidase (SA-β-gal) staining, p53 and p21^Cip1/WAF1 expression, and reactive oxygen species (ROS) production in proliferating IMR-90 cells. CK2α over-expression almost abolished this event. Taken together, the present results suggest that the upregulation of miR-760 and miR-186 is associated with replicative senescence in human lung fibroblast cells, and their cooperative action with miR-337-3p and miR-216b may induce replicative senescence through CK2α downregulation-dependent ROS generation.     

Expression of cellular genes in HPV16-immortalized and cigarette smoke condensate-transformed human endocervical cells

We studied the molecular mechanism of successive multistep cervical carcinogenic progression with our previously established in vitro model system. This system was composed of primary human endocervical cells (HEN), two lines of HEN immortalized by HPV16 and their counterparts subsequently malignantly transformed by cigarette smoke condensate (CSC). The expression was examined of diverse cellular genes associated with oncogenesis and senescence, especially for cervical cancer. Consistent results were seen for the pairs of immortalized and malignantly transformed lines. Immortalization of HEN by HPV16 resulted in enhanced expression of H-ras, c-myc, B-myb, p53, p16^INK4 and PCNA mRNA; enhanced expression of p16 and PCNA proteins; decreased expression of WAF1/p21/Cip1/Sid1 and fibronectin mRNA; and decreased p53 protein. On the other hand, the CSC-transformed counterparts of HPV16-immortalized cells had up-regulated levels of B-myb, p53 and WAF1 mRNA and p53 protein. Our results indicate that the differential activation or inactivation of multiple cellular genes is important for the immortalization, as well as the transformation, of human cervical cells. Further, we suggest that our in vitro model system is useful for investigating the molecular mechanism of multistep cervical carcinogenesis. J. Cell. Biochem. 66: 309–321, 1997. © 1997 Wiley-Liss, Inc.     

The Epstein-Barr virus (EBV)-induced tumor suppressor microRNA MiR-34a is growth promoting in EBV-infected B cells

Epstein-Barr virus (EBV) infection of primary human B cells drives their indefinite proliferation into lymphoblastoid cell lines (LCLs). B cell immortalization depends on expression of viral latency genes, as well as the regulation of host genes. Given the important role of microRNAs (miRNAs) in regulating fundamental cellular processes, in this study, we assayed changes in host miRNA expression during primary B cell infection by EBV. We observed and validated dynamic changes in several miRNAs from early proliferation through immortalization; oncogenic miRNAs were induced, and tumor suppressor miRNAs were largely repressed. However, one miRNA described as a p53-targeted tumor suppressor, miR-34a, was strongly induced by EBV infection and expressed in many EBV and Kaposi's sarcoma-associated herpesvirus (KSHV)-infected lymphoma cell lines. EBV latent membrane protein 1 (LMP1) was sufficient to induce miR-34a requiring downstream NF-κB activation but independent of functional p53. Furthermore, overexpression of miR-34a was not toxic in several B lymphoma cell lines, and inhibition of miR-34a impaired the growth of EBV-transformed cells. This study identifies a progrowth role for a tumor-suppressive miRNA in oncogenic-virus-mediated transformation, highlighting the importance of studying miRNA function in different cellular contexts.     

MiR-29a inhibits cell proliferation and induces cell cycle arrest through the downregulation of p42.3 in human gastric cancer

As a newly identified and characterized gene, p42.3 is associated with cell proliferation and tumorigenicity. The expression of p42.3 is upregulated in human gastric cancer (GC), but its underlying mechanisms of action are not well understood. MicroRNAs (miRNAs) are known to play vital regulatory roles in many cellular processes. Here we utilized bioinformatics and experimental approaches to investigate the regulatory relationship between miRNAs and the p42.3 gene. We showed that miR-29a could repress p42.3 expression at both the mRNA and protein levels via directly binding to its 3'UTR. Furthermore, an inverse relationship was observed between miR-29a and p42.3 expression in gastric cancer cell lines and GC tissue samples, especially in cases where p42.3 was downregulated. Taken together, we have elucidated previously unrecognized roles of miR-29a and indicated that miR-29a may function, at least partially, by targeting the p42.3 gene in human GC.     

Differentiation Induces Dramatic Changes in miRNA Profile,Where Loss of Dicer Diverts Differentiating SH-SY5Y Cells Toward Senescence

MicroRNAs (miRNAs) are generated by endonuclease activity of Dicer, which also helps in loading of miRNAs to their target sequences. SH-SY5Y, a human neuroblastoma and a cellular model of neurodevelopment, consistently expresses genes related to neurodegenerative disorders at different biological levels (DNA, RNA, and proteins). Using SH-SY5Y cells, we have studied the role of Dicer and miRNAs in neuronal differentiation and explored involvement of P53, a master regulator of gene expression in differentiation-induced induction of miRNAs. Knocking down Dicer gene induced senescence in differentiating SH-SY5Y cells, which indicate the essential role of Dicer in brain development. Differentiation of SH-SY5Y cells by retinoic acid (RA) or RA + brain-derived neurotrophic factor (BDNF) induced dramatic changes in global miRNA expression. Fully differentiated SH-SY5Y cells (5-day RA followed by 3-day BDNF) significantly (p < 0.05 and atleast >3-fold change) upregulated and downregulated the expression of 77 and 17 miRNAs, respectively. Maximum increase was observed in the expression of miR-193-5p, miR-199a-5p, miR-192, miR-145, miR-28-5p, miR-29b, and miR-222 after RA exposure and miR-193-5p, miR-146a, miR-21, miR-199a-5p, miR-153, miR-29b, and miR-222 after RA + BDNF exposure in SH-SY5Y cells. Exploring the role of P53 in differentiating SH-SY5Y cells, we have observed that induction of miR-222, miR-192, and miR-145 is P53 dependent and expression of miR-193a-5p, miR-199a-5p, miR-146a, miR-21, miR-153, and miR-29b is P53 independent. In conclusion, decreased Dicer level enforces differentiating cells to senescence, and differentiating SH-SY5Y cells needs increased expression of P53 to cope up with changes in protein levels of mature neurons.     

Upregulated microRNA-29a by hepatitis B virus X protein enhances hepatoma cell migration by targeting PTEN in cell culture model

Hepatitis B virus X protein (HBx) plays important roles in the development of hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) contribute to cancer development by acting as oncogenes or tumor suppressors. Previously, we reported that HBx was able to promote the migration of hepatoma HepG2 cells. However, the regulation of miRNAs in the development of HBV-related HCC is poorly understood. In the present study, we reported that miR-29a was a novel regulator of migration of hepatoma cells mediated by HBx. Our data showed that the expression of miR-29a was dramatically increased in p21-HBx transgenic mice, HBx-transfected hepatoma HepG2-X (or H7402-X) cells and HepG2.2.15 cells that constitutively replicate HBV. However, our data showed that miR-29a was upregulated in 4 of the 11 clinical HCC samples. We found that the overexpression of miR-29a promoted the migration of HepG2 cells, while a specific miR-29a inhibitor could partially abolish the enhanced migration of HepG2-X cells. Moreover, we identified PTEN was one of the target genes of miR-29a in HepG2 cells. The deletion of the miR-29a-binding site was able to abolish the role of miR-29a in suppression of luciferase activity of the PTEN 3'UTR reporter. Meanwhile, the overexpression of PTEN was able to reverse the promoted migration of HepG2 cells mediated by miR-29a. Moreover, our data showed that the modulation of Akt phosphorylation, a downstream factor of PTEN, was involved in the cell migration enhanced by miR-29a, suggesting that miR-29a is responsible for the cell migration through its target gene PTEN. Thus, we conclude that miR-29a is involved in the regulation of migration of hepatoma cells mediated by HBx through PTEN in cell culture model.