scholarly journals Events in the immortalizing process of primary human mammary epithelial cells by the catalytic subunit of human telomerase

2002 ◽  
Vol 365 (3) ◽  
pp. 765-772 ◽  
Author(s):  
Hyunggee KIM ◽  
James FARRIS ◽  
Shelly A. CHRISTMAN ◽  
Byung-Whi KONG ◽  
Linda K. FOSTER ◽  
...  
Keyword(s):  
Single Copy ◽  
Telomerase Activity ◽  
Genetic Alterations ◽  
Mammary Epithelial ◽  
Catalytic Subunit ◽  
Early Passage ◽  
Htert Gene ◽  
Human Mammary Epithelial ◽  
Human Telomerase

The in vitro immortalization of primary human mammary epithelial (HME) cells solely by the exogenous introduction of the catalytic subunit of human telomerase (hTERT) has been achieved. Early passage hTERT-transfected HME (T-HME) cells continuously decreased the length and density of telomeres even in the presence of telomerase activity, with a significant number of cells staining positive for senescence-associated β-galactosidase (SA-β-gal). Subsequently, with the increase in cell passages, the copy number of the exogenously transfected hTERT gene and the percentage of SA-β-gal positive cells were found to decrease. Eventually, a single copy of the exogenous hTERT gene was observed in the relatively later passage T-HME cells in which telomere length was elongated and stabilized without obvious activation of endogenous hTERT and c-Myc expression. In T-HME cells, the expression of two p53 regulated genes p21WAF and HDM2 increased (as in primary senescent HME cells), and was found to be further elevated as the function of p53 was activated by treatment with DNA-damaging agents. p16INK4a was shown to be significantly higher in the primary senescent HME and the early passage T-HME cells when compared with the primary presenescent HME cells, with a dramatic repression of p16INK4a observed in the later passage T-HME cells. In addition, the expression of E2F1 and its transcription factor activity were found to be significantly higher in the later passage T-HME cells when compared with the earlier passage T-HME cells. Together, our results indicate that in vitro immortalization in HME cells may require the activation of the function of telomerase and other genetic alterations such as the spontaneous loss of p16INK4a expression.

scholarly journals Gradual Phenotypic Conversion Associated with Immortalization of Cultured Human Mammary Epithelial Cells

1997 ◽  
Vol 8 (12) ◽  
pp. 2391-2405 ◽  
Author(s):  
Martha R. Stampfer ◽  
Andrea Bodnar ◽  
James Garbe ◽  
Michelle Wong ◽  
Alison Pan ◽  
...  
Keyword(s):  
Transforming Growth Factor ◽  
Telomerase Activity ◽  
Mammary Epithelial ◽  
Good Growth ◽  
Activity Levels ◽  
Early Passage ◽  
Mass Cultures ◽  
Human Mammary Epithelial

Examination of the process of immortal transformation in early passages of two human mammary epithelial cell (HMEC) lines suggests the involvement of an epigenetic step. These lines, 184A1 and 184B5, arose after in vitro exposure of finite lifespan 184 HMEC to a chemical carcinogen, and both are clonally derived. Although early-passage mass cultures of 184A1 and 184B5 maintained continuous slow growth, most individual cells lost proliferative ability. Uniform good growth did not occur until 20–30 passages after the lines first appeared. Early-passage cultures expressed little or no telomerase activity and telomeres continued to shorten with increasing passage. Telomerase activity was first detected when the telomeres became critically short, and activity levels gradually increased thereafter. Early-passage cultures had little or no ability to maintain growth in transforming growth factor-β (TGFβ); however, both mass cultures and clonal isolates showed a very gradual increase in the number of cells displaying progressively increased ability to maintain growth in TGFβ. A strong correlation between capacity to maintain growth in the presence of TGFβ and expression of telomerase activity was observed. We have used the term “conversion” to describe this process of gradual acquisition of increased growth capacity in the absence or presence of TGFβ and reactivation of telomerase. We speculate that the development of extremely short telomeres may result in gradual, epigenetic-based changes in gene expression. Understanding the underlying mechanisms of HMEC conversion in vitro may provide new insight into the process of carcinogenic progression in vivo and offer novel modes for therapeutic intervention.

scholarly journals Apoptotic endonuclease EndoG regulates alternative splicing of human telomerase catalytic subunit hTERT

2016 ◽  
Vol 62 (5) ◽  
pp. 544-554 ◽  
Author(s):  
D.D. Zhdanov ◽  
D.A. Vasina ◽  
E.V. Orlova ◽  
V.S. Orlova ◽  
M.V. Pokrovskaya ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Telomerase Activity ◽  
Catalytic Subunit ◽  
Over Expression ◽  
Htert Gene ◽  
Template Strand ◽  
Non Coding Rna ◽  
Dna Strand ◽  
Human Telomerase ◽  
Long Non Coding Rna

Human telomerase catalytic subunit hTERT is subjected to alternative splicing results in loss of its function and leads to decrease of telomerase activity. However, very little is known about the mechanism of hTERT pre-mRNA alternative splicing. Apoptotic endonuclease EndoG is known to participate this process. The aim of this study was to determine the role of EndoG in regulation of hTERT alternative splicing. Increased expression of b-deletion splice variant was determined during EndoG over-expression in CaCo-2 cell line, after EndoG treatment of cell cytoplasm and nuclei and after nuclei incubation with EndoG digested cell RNA. hTERT alternative splicing was induced by 47-mer RNA oligonucleotide in naked nuclei and in cells after transfection. Identified long non-coding RNA, that is the precursor of 47-mer RNA oligonucleotide. Its size is 1754 nucleotides. Based on the results the following mechanism was proposed. hTERT pre-mRNA is transcribed from coding DNA strand while long non-coding RNA is transcribed from template strand of hTERT gene. EndoG digests long non-coding RNA and produces 47-mer RNA oligonucleotide complementary to hTERT pre-mRNA exon 8 and intron 8 junction place. Interaction of 47-mer RNA oligonucleotide and hTERT pre-mRNA causes alternative splicing.

scholarly journals Induction of hTERT Expression and Telomerase Activity by Estrogens in Human Ovary Epithelium Cells

2000 ◽  
Vol 20 (11) ◽  
pp. 3764-3771 ◽  
Author(s):  
Silvia Misiti ◽  
Simona Nanni ◽  
Giulia Fontemaggi ◽  
Yu-Sheng Cong ◽  
Jianping Wen ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Molecular Mechanisms ◽  
Estrogen Receptor Α ◽  
Telomerase Activity ◽  
Human Ovary ◽  
Htert Gene ◽  
Human Telomerase

ABSTRACT In mammals, molecular mechanisms and factors involved in the tight regulation of telomerase expression and activity are still largely undefined. In this study, we provide evidence for a role of estrogens and their receptors in the transcriptional regulation of hTERT, the catalytic subunit of human telomerase and, consequently, in the activation of the enzyme. Through a computer analysis of the hTERT 5′-flanking sequences, we identified a putative estrogen response element (ERE) which was capable of binding in vitro human estrogen receptor α (ERα). In vivo DNA footprinting revealed specific modifications of the ERE region in ERα-positive but not ERα-negative cells upon treatment with 17β-estradiol (E2), indicative of estrogen-dependent chromatin remodelling. In the presence of E2, transient expression of ERα but not ERβ remarkably increased hTERT promoter activity, and mutation of the ERE significantly reduced this effect. No telomerase activity was detected in human ovary epithelial cells grown in the absence of E2, but the addition of the hormone induced the enzyme within 3 h of treatment. The expression of hTERT mRNA and protein was induced in parallel with enzymatic activity. This prompt estrogen modulation of telomerase activity substantiates estrogen-dependent transcriptional regulation of the hTERT gene. The identification of hTERT as a target of estrogens represents a novel finding which advances the understanding of telomerase regulation in hormone-dependent cells and has implications for a potential role of hormones in their senescence and malignant conversion.

scholarly journals Functional Regions of Human Telomerase Reverse Transcriptase and Human Telomerase RNA Required for Telomerase Activity and RNA-Protein Interactions

2001 ◽  
Vol 21 (5) ◽  
pp. 1888-1897 ◽  
Author(s):  
François Bachand ◽  
Chantal Autexier
Keyword(s):  
Reverse Transcriptase ◽  
Rna Binding ◽  
Telomerase Activity ◽  
Catalytic Subunit ◽  
Telomerase Rna ◽  
Dna Repeats ◽  
Human Telomerase Reverse Transcriptase ◽  
Template Sequence ◽  
Human Telomerase

ABSTRACT Telomerase is a specialized reverse transcriptase (RT) that is minimally composed of a protein catalytic subunit and an RNA component. The RNA subunit contains a short template sequence that directs the synthesis of DNA repeats at the ends of chromosomes. Human telomerase activity can be reconstituted in vitro by the expression of the human telomerase protein catalytic subunit (hTERT) in the presence of recombinant human telomerase RNA (hTR) in a rabbit reticulocyte lysate (RRL) system. We analyzed telomerase activity and binding of hTR to hTERT in RRL by expressing different hTERT and hTR variants. hTRs containing nucleotide substitutions that are predicted to disrupt base pairing in the P3 helix of the pseudoknot weakly reconstituted human telomerase activity yet retained their ability to bind hTERT. Our results also identified two distinct regions of hTR that can independently bind hTERT in vitro. Furthermore, sequences or structures between nucleotides 208 and 330 of hTR (which include the conserved CR4-CR5 domain) were found to be important for hTERT-hTR interactions and for telomerase activity reconstitution. Human TERT carboxy-terminal amino acid deletions extending to motif E or the deletion of the first 280 amino acids abolished human telomerase activity without affecting the ability of hTERT to associate with hTR, suggesting that the RT and RNA binding functions of hTERT are separable. These results indicate that the reconstitution of human telomerase activity in vitro requires regions of hTERT that (i) are distinct from the conserved RT motifs and (ii) bind nucleotides distal to the hTR template sequence.

scholarly journals Hypermethylation of the human telomerase catalytic subunit (hTERT) gene correlates with telomerase activity

2002 ◽  
Vol 101 (4) ◽  
pp. 335-341 ◽  
Author(s):  
Isabelle Guilleret ◽  
Pu Yan ◽  
Fabienne Grange ◽  
Richard Braunschweig ◽  
Fred T. Bosman ◽  
...  
Keyword(s):  
Telomerase Activity ◽  
Catalytic Subunit ◽  
Htert Gene ◽  
Human Telomerase

scholarly journals Two Inactive Fragments of the Integral RNA Cooperate To Assemble Active Telomerase with the Human Protein Catalytic Subunit (hTERT) In Vitro

1999 ◽  
Vol 19 (9) ◽  
pp. 6207-6216 ◽  
Author(s):  
Valerie M. Tesmer ◽  
Lance P. Ford ◽  
Shawn E. Holt ◽  
Bryan C. Frank ◽  
Xiaoming Yi ◽  
...  
Keyword(s):  
De Novo ◽  
Telomerase Activity ◽  
Catalytic Subunit ◽  
Human Protein ◽  
Cell Extracts ◽  
In Vitro Assembly ◽  
Reticulocyte Lysates ◽  
Minimal Sequence ◽  
Human Telomerase

ABSTRACT We have mapped the 5′ and 3′ boundaries of the region of the human telomerase RNA (hTR) that is required to produce activity with the human protein catalytic subunit (hTERT) by using in vitro assembly systems derived from rabbit reticulocyte lysates and human cell extracts. The region spanning nucleotides +33 to +325 of the 451-base hTR is the minimal sequence required to produce levels of telomerase activity that are comparable with that made with full-length hTR. Our results suggest that the sequence approximately 270 bases downstream of the template is required for efficient assembly of active telomerase in vitro; this sequence encompasses a substantially larger portion of the 3′ end of hTR than previously thought necessary. In addition, we identified two fragments of hTR (nucleotides +33 to +147 and +164 to +325) that cannot produce telomerase activity when combined separately with hTERT but can function together to assemble active telomerase. These results suggest that the minimal sequence of hTR can be divided into two sections, both of which are required for de novo assembly of active telomerase in vitro.

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