scholarly journals EML4-ALK induces cellular senescence in mortal normal human cells and promotes anchorage-independent growth in hTERT-transduced normal human cells

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Akihiko Miyanaga ◽  
Masaru Matsumoto ◽  
Jessica A. Beck ◽  
Izumi Horikawa ◽  
Takahiro Oike ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cellular Senescence ◽  
Human Fibroblasts ◽  
Human Cells ◽  
Functional Study ◽  
Sequencing Analysis ◽  
Anchorage Independent Growth ◽  
Normal Human ◽  
Normal Human Cells

Abstract Background Chromosomal inversions involving anaplastic lymphoma kinase (ALK) and echinoderm microtubule associated protein like 4 (EML4) generate a fusion protein EML4-ALK in non-small cell lung cancer (NSCLC). The understanding of EML4-ALK function can be improved by a functional study using normal human cells. Methods Here we for the first time conduct such study to examine the effects of EML4-ALK on cell proliferation, cellular senescence, DNA damage, gene expression profiles and transformed phenotypes. Results The lentiviral expression of EML4-ALK in mortal, normal human fibroblasts caused, through its constitutive ALK kinase activity, an early induction of cellular senescence with accumulated DNA damage, upregulation of p16INK4A and p21WAF1, and senescence-associated β-galactosidase (SA-β-gal) activity. In contrast, when EML4-ALK was expressed in normal human fibroblasts transduced with telomerase reverse transcriptase (hTERT), which is activated in the vast majority of NSCLC, the cells showed accelerated proliferation and acquired anchorage-independent growth ability in soft-agar medium, without accumulated DNA damage, chromosome aberration, nor p53 mutation. EML4-ALK induced the phosphorylation of STAT3 in both mortal and hTERT-transduced cells, but RNA sequencing analysis suggested that the different signaling pathways contributed to the different phenotypic outcomes in these cells. While EML4-ALK also induced anchorage-independent growth in hTERT-immortalized human bronchial epithelial cells in vitro, the expression of EML4-ALK alone did not cause detectable in vivo tumorigenicity in immunodeficient mice. Conclusions Our data indicate that the expression of hTERT is critical for EML4-ALK to manifest its in vitro transforming activity in human cells. This study provides the isogenic pairs of human cells with and without EML4-ALK expression.

scholarly journals EML4-ALK induces cellular senescence in mortal normal human cells and promotes anchorage-independent growth in hTERT-transduced normal human cells

2020 ◽  
Author(s):  
Akihiko Miyanaga ◽  
Masaru Matsumoto ◽  
Jessica A. Beck ◽  
Izumi Horikawa ◽  
Takahiro Oike ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cellular Senescence ◽  
Human Fibroblasts ◽  
Human Cells ◽  
Functional Study ◽  
Sequencing Analysis ◽  
Anchorage Independent Growth ◽  
Normal Human ◽  
Normal Human Cells

Abstract Background: Chromosomal inversions involving anaplastic lymphoma kinase (ALK) and echinoderm microtubule associated protein like 4 (EML4) generate a fusion protein EML4-ALK in non-small cell lung cancer (NSCLC). The understanding of EML4-ALK function can be improved by a functional study using normal human cells.Methods: Here we for the first time conduct such study to examine the effects of EML4-ALK on cell proliferation, cellular senescence, DNA damage, gene expression profiles and transformed phenotypes.Results: The lentiviral expression of EML4-ALK in mortal, normal human fibroblasts caused, through its constitutive ALK kinase activity, an early induction of cellular senescence with accumulated DNA damage, upregulation of p16INK4A and p21WAF1, and senescence-associated b-galactosidase (SA-β-gal) activity. In contrast, when EML4-ALK was expressed in normal human fibroblasts transduced with telomerase reverse transcriptase (hTERT), which is activated in the vast majority of NSCLC, the cells showed accelerated proliferation and acquired anchorage-independent growth ability in soft-agar medium, without accumulated DNA damage, chromosome aberration, nor p53 mutation. EML4-ALK induced the phosphorylation of STAT3 in both mortal and hTERT-transduced cells, but RNA sequencing analysis suggested that the different signaling pathways contributed to the different phenotypic outcomes in these cells. While EML4-ALK also induced anchorage-independent growth in hTERT-immortalized human bronchial epithelial cells in vitro, the expression of EML4-ALK alone did not cause detectable in vivo tumorigenicity in immunodeficient mice.Conclusions: Our data indicate that the expression of hTERT is critical for EML4-ALK to manifest its in vitro transforming activity in human cells. This study provides the isogenic pairs of human cells with and without EML4-ALK expression.

scholarly journals EML4-ALK induces cellular senescence in mortal normal human cells and promotes anchorage-independent growth in immortalized normal human cells

2020 ◽  
Author(s):  
Akihiko Miyanaga ◽  
Masaru Matsumoto ◽  
Jessica A. Beck ◽  
Izumi Horikawa ◽  
Takahiro Oike ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cellular Senescence ◽  
Human Fibroblasts ◽  
Human Cells ◽  
Functional Study ◽  
Sequencing Analysis ◽  
Anchorage Independent Growth ◽  
Normal Human ◽  
Normal Human Cells

Abstract Background: Chromosomal inversions involving anaplastic lymphoma kinase (ALK) and echinoderm microtubule associated protein like 4 (EML4) generate a fusion protein EML4-ALK in non-small cell lung cancer (NSCLC). The understanding of EML4-ALK function can be improved by a functional study using normal human cells.Methods: Here we for the first time conduct such study to examine the effects of EML4-ALK on cell proliferation, cellular senescence, DNA damage, gene expression profiles and transformed phenotypes.Results: The lentiviral expression of EML4-ALK in mortal, normal human fibroblasts caused, through its constitutive ALK kinase activity, an early induction of cellular senescence with senescence-associated b-galactosidase activity, upregulation of p16INK4A and p21WAF1, and accumulated DNA damage. In contrast, when EML4-ALK was expressed in normal human fibroblasts immortalized by telomerase reverse transcriptase (hTERT), which is activated in the vast majority of NSCLC, the cells showed accelerated proliferation and acquired anchorage-independent growth ability in soft-agar medium, without accumulated DNA damage, chromosome aberration, nor p53 mutation. EML4-ALK induced the phosphorylation of STAT3 in both mortal and immortalized cells, but RNA sequencing analysis suggested that the different STAT3-regulated signaling pathways contributed to the different phenotypic outcomes in these cells. While EML4-ALK also induced anchorage-independent growth in hTERT-immortalized human bronchial epithelial cells in vitro, the expression of EML4-ALK alone did not cause detectable in vivo tumorigenicity in immunodeficient mice.Conclusions: Our data indicate that the immortalized state is critical for EML4-ALK to manifest its in vitro transforming activity in human cells. This study provides the isogenic pairs of human cells with and without EML4-ALK expression.

scholarly journals EML4-ALK induces cellular senescence in mortal normal human cells and promotes anchorage-independent growth in hTERT-transduced normal human cells

2020 ◽  
Author(s):  
Akihiko Miyanaga ◽  
Masaru Matsumoto ◽  
Jessica A. Beck ◽  
Izumi Horikawa ◽  
Takahiro Oike ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cellular Senescence ◽  
Human Fibroblasts ◽  
Human Cells ◽  
Functional Study ◽  
Sequencing Analysis ◽  
Anchorage Independent Growth ◽  
Normal Human ◽  
Normal Human Cells

Abstract Background: Chromosomal inversions involving anaplastic lymphoma kinase (ALK) and echinoderm microtubule associated protein like 4 (EML4) generate a fusion protein EML4-ALK in non-small cell lung cancer (NSCLC). The understanding of EML4-ALK function can be improved by a functional study using normal human cells.Methods: Here we for the first time conduct such study to examine the effects of EML4-ALK on cell proliferation, cellular senescence, DNA damage, gene expression profiles and transformed phenotypes.Results: The lentiviral expression of EML4-ALK in mortal, normal human fibroblasts caused, through its constitutive ALK kinase activity, an early induction of cellular senescence with accumulated DNA damage, upregulation of p16INK4A and p21WAF1, and senescence-associated b-galactosidase (SA-β-gal) activity. In contrast, when EML4-ALK was expressed in normal human fibroblasts transduced with telomerase reverse transcriptase (hTERT), which is activated in the vast majority of NSCLC, the cells showed accelerated proliferation and acquired anchorage-independent growth ability in soft-agar medium, without accumulated DNA damage, chromosome aberration, nor p53 mutation. EML4-ALK induced the phosphorylation of STAT3 in both mortal and hTERT-transduced cells, but RNA sequencing analysis suggested that the different signaling pathways contributed to the different phenotypic outcomes in these cells. While EML4-ALK also induced anchorage-independent growth in hTERT-immortalized human bronchial epithelial cells in vitro, the expression of EML4-ALK alone did not cause detectable in vivo tumorigenicity in immunodeficient mice.Conclusions: Our data indicate that the expression of hTERT is critical for EML4-ALK to manifest its in vitro transforming activity in human cells. This study provides the isogenic pairs of human cells with and without EML4-ALK expression.

scholarly journals Reduction of X-ray-induced DNA damage in normal human cells treated with the PrC-210 radioprotector

Biology Open ◽  
2018 ◽  
Vol 7 (10) ◽  
pp. bio035113 ◽  
Author(s):  
Michael Brand ◽  
Matthias Sommer ◽  
Frank Jermusek ◽  
William E. Fahl ◽  
Michael Uder
Keyword(s):  
Dna Damage ◽  
Human Cells ◽  
X Ray ◽  
Normal Human ◽  
Normal Human Cells

scholarly journals Adenovirus E1B 55-Kilodalton Protein Is Required for both Regulation of mRNA Export and Efficient Entry into the Late Phase of Infection in Normal Human Fibroblasts

2006 ◽  
Vol 80 (2) ◽  
pp. 964-974 ◽  
Author(s):  
Ramon Gonzalez ◽  
Wenying Huang ◽  
Renee Finnen ◽  
Courtney Bragg ◽  
S. J. Flint
Keyword(s):  
Dna Synthesis ◽  
Hela Cells ◽  
Human Fibroblasts ◽  
Late Phase ◽  
Mrna Export ◽  
Human Cells ◽  
Insertion Mutation ◽  
Viral Dna ◽  
Normal Human ◽  
Normal Human Cells

ABSTRACT The human adenovirus type 5 (Ad5) E1B 55-kDa protein is required for selective nuclear export of viral late mRNAs from the nucleus and concomitant inhibition of export of cellular mRNAs in HeLa cells and some other human cell lines, but its contributions(s) to replication in normal human cells is not well understood. We have therefore examined the phenotypes exhibited by viruses carrying mutations in the E1B 55-kDa protein coding sequence in normal human fibroblast (HFFs). Ad5 replicated significantly more slowly in HFFs than it does in tumor cells, a difference that is the result of delayed entry into the late phase of infection. The A143 mutation, which specifically impaired export of viral late mRNAs from the nucleus in infected HeLa cells (R. A. Gonzalez and S. J. Flint, J. Virol. 76:4507-4519, 2002), induced a more severe defect in viral mRNA export in HFFs. This observation indicates that the E1B 55-kDa protein regulates mRNA export during the late phase of infection of normal human cells. Other mutants exhibited phenotypes not observed in HeLa cells. In HFFs infected by the null mutant Hr6, synthesis of viral late mRNAs and proteins was severely impaired. Such defects in late gene expression were the result of inefficient progression into the late phase of infection, for viral DNA synthesis was 10-fold less efficient in Hr6-infected HFFs than in cells infected by Ad5. Similar, but less severe, defects in viral DNA synthesis were induced by the insertion mutation H224, which has been reported to inhibit binding of the E1B 55-kDa protein to p53 (C. C. Kao, P. R. Yew, and A. J. Berk, Virology 179:806-814, 1990).

scholarly journals Significant Suppression of CT Radiation-Induced DNA Damage in Normal Human Cells by the PrC-210 Radioprotector

2018 ◽  
Vol 190 (2) ◽  
pp. 133 ◽  
Author(s):  
Frank Jermusek ◽  
Chelsea Benedict ◽  
Emma Dreischmeier ◽  
Michael Brand ◽  
Michael Uder ◽  
...  
Keyword(s):  
Dna Damage ◽  
Human Cells ◽  
Significant Suppression ◽  
Ct Radiation ◽  
Radiation Induced ◽  
Normal Human ◽  
Normal Human Cells

scholarly journals Supra-physiological folic acid concentrations induce aberrant DNA methylation in normal human cells in vitro

Epigenetics ◽  
2012 ◽  
Vol 7 (7) ◽  
pp. 689-694 ◽  
Author(s):  
Michelle A. Charles ◽  
Ian T. Johnson ◽  
Nigel J. Belshaw
Keyword(s):  
Dna Methylation ◽  
Folic Acid ◽  
Human Cells ◽  
Normal Human ◽  
Normal Human Cells ◽  
Aberrant Dna Methylation

scholarly journals Analysis of Genomic Integrity and p53-Dependent G1Checkpoint in Telomerase-Induced Extended-Life-Span Human Fibroblasts

1999 ◽  
Vol 19 (3) ◽  
pp. 2373-2379 ◽  
Author(s):  
Homayoun Vaziri ◽  
Jeremy A. Squire ◽  
Tej K. Pandita ◽  
Grace Bradley ◽  
Robert M. Kuba ◽  
...  
Keyword(s):  
Ionizing Radiation ◽  
Life Span ◽  
P53 Protein ◽  
Human Fibroblasts ◽  
Strand Break ◽  
Human Cells ◽  
Normal Human ◽  
Normal Human Cells ◽  
Extended Life

ABSTRACT Life span determination in normal human cells may be regulated by nucleoprotein structures called telomeres, the physical ends of eukaryotic chromosomes. Telomeres have been shown to be essential for chromosome stability and function and to shorten with each cell division in normal human cells in culture and with age in vivo. Reversal of telomere shortening by the forced expression of telomerase in normal cells has been shown to elongate telomeres and extend the replicative life span (H. Vaziri and S. Benchimol, Curr. Biol. 8:279–282, 1998; A. G. Bodnar et al., Science 279:349–352, 1998). Extension of the life span as a consequence of the functional inactivation of p53 is frequently associated with loss of genomic stability. Analysis of telomerase-induced extended-life-span fibroblast (TIELF) cells by G banding and spectral karyotyping indicated that forced extension of the life span by telomerase led to the transient formation of aberrant structures, which were subsequently resolved in higher passages. However, the p53-dependent G1 checkpoint was intact as assessed by functional activation of p53 protein in response to ionizing radiation and subsequent p53-mediated induction of p21Waf1/Cip1/Sdi1. TIELF cells were not tumorigenic and had a normal DNA strand break rejoining activity and normal radiosensitivity in response to ionizing radiation.

scholarly journals Human RECQL5 participates in the removal of endogenous DNA damage

2012 ◽  
Vol 23 (21) ◽  
pp. 4273-4285 ◽  
Author(s):  
Takashi Tadokoro ◽  
Mahesh Ramamoorthy ◽  
Venkateswarlu Popuri ◽  
Alfred May ◽  
Jingyan Tian ◽  
...  
Keyword(s):  
Dna Damage ◽  
Genome Stability ◽  
Excision Repair ◽  
Human Cells ◽  
Recq Helicase ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Normal Human ◽  
Normal Human Cells ◽  
Base Excision

Human RECQL5 is a member of the RecQ helicase family, which maintains genome stability via participation in many DNA metabolic processes, including DNA repair. Human cells lacking RECQL5 display chromosomal instability. We find that cells depleted of RECQL5 are sensitive to oxidative stress, accumulate endogenous DNA damage, and increase the cellular poly(ADP-ribosyl)ate response. In contrast to the RECQ helicase family members WRN, BLM, and RECQL4, RECQL5 accumulates at laser-induced single-strand breaks in normal human cells. RECQL5 depletion affects the levels of PARP-1 and XRCC1, and our collective results suggest that RECQL5 modulates and/or directly participates in base excision repair of endogenous DNA damage, thereby promoting chromosome stability in normal human cells.

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