Alterations of DNA content in human endometrial stromal cells transfected with a temperature-sensitive SV40: tetraploidization and physiological consequences

Rinehart, Clifford A.; Mayben, John P.; Butler, Thomas D; Haskill, J. S.; Kaufman, David G.

doi:10.1093/carcin/13.1.63

Cited by 12 publications

(7 citation statements)

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“…We reasoned that the best view on authentic genome and transcript changes after dual CRISPR cleavage would be obtained in cells with only one target chromosome, without the possibility of target competition or gene conversion between DMPK alleles of different lengths, as might occur in patient cells with two chromosomes 19. Unfortunately, this criterion was not fully met because the DM500 myoblast lineage had undergone tetraploidization during propagation in culture at the permissive temperature (Figure S2), a common feature of cells that express the SV40 large T antigen during cell cycle progression 39, 40. Small-pool PCR analysis of repeat length variation in the DM500 cell population revealed three types of main alleles with ∼540, ∼570, and ∼610 CTG⋅CAG repeats (Figures 3A–3D) and a subset of rarer expansion and contraction events around these two alleles.…”

Section: Resultsmentioning

confidence: 99%

CRISPR/Cas9-Induced (CTG⋅CAG) n Repeat Instability in the Myotonic Dystrophy Type 1 Locus: Implications for Therapeutic Genome Editing

et al. 2017

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Myotonic dystrophy type 1 (DM1) is caused by (CTG⋅CAG)n-repeat expansion within the DMPK gene and thought to be mediated by a toxic RNA gain of function. Current attempts to develop therapy for this disease mainly aim at destroying or blocking abnormal properties of mutant DMPK (CUG)n RNA. Here, we explored a DNA-directed strategy and demonstrate that single clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-cleavage in either its 5′ or 3′ unique flank promotes uncontrollable deletion of large segments from the expanded trinucleotide repeat, rather than formation of short indels usually seen after double-strand break repair. Complete and precise excision of the repeat tract from normal and large expanded DMPK alleles in myoblasts from unaffected individuals, DM1 patients, and a DM1 mouse model could be achieved at high frequency by dual CRISPR/Cas9-cleavage at either side of the (CTG⋅CAG)n sequence. Importantly, removal of the repeat appeared to have no detrimental effects on the expression of genes in the DM1 locus. Moreover, myogenic capacity, nucleocytoplasmic distribution, and abnormal RNP-binding behavior of transcripts from the edited DMPK gene were normalized. Dual sgRNA-guided excision of the (CTG⋅CAG)n tract by CRISPR/Cas9 technology is applicable for developing isogenic cell lines for research and may provide new therapeutic opportunities for patients with DM1.

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Section: Resultsmentioning

confidence: 99%

CRISPR/Cas9-Induced (CTG⋅CAG) n Repeat Instability in the Myotonic Dystrophy Type 1 Locus: Implications for Therapeutic Genome Editing

et al. 2017

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“…5). We note that SV40 large T antigen, which binds and inactivates pRb as well as other cellular proteins, induces tetraploidy in certain cell types (Ornitz et al 1987;Kuhar and Lehman 1991;Rinehart et al 1992Rinehart et al , 1993. In addition, exocrine tissues of E2F1 -/ -mice contain cells with abnormally large nuclei and binucleated cells (Field et al 1996;Yamasaki et al 1996).…”

Section: Genes and Developmentmentioning

confidence: 86%

pRb controls proliferation, differentiation, and death of skeletal muscle cells and other lineages during embryogenesis.

Zacksenhaus

Jiang²,

Chung³

et al. 1996

Genes Dev.

282

254

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Mice deficient for the RB gene (RB-/-), prior to death at embryonic day 14.5, show increased cell death in all tissues that normally express RBI: the nervous system, liver, lens, and skeletal muscle precursor cells. We have generated transgenic mice (RBlox) that express low levels of pRb, driven by an RB1 minigene. RBIox/RB -/-mutant fetuses die at birth with specific skeletal muscle defects, including increased cell death prior to myoblast fusion, shorter myotubes with fewer myofibrils, reduced muscle fibers, accumulation of elongated nuclei that actively synthesized DNA within the myotubes, and reduction in expression of the late muscle-specific genes MCK and MRF4. Thus, insufficient pRb results in failure of myogenesis in vivo, manifest in two ways. First, the massive apoptosis of myoblasts implicates a role of pRb in cell survival. Second, surviving myotubes failed to develop normally and accumulated large polyploid nuclei, implicating pRb in permanent withdrawal from the cell cycle. These results demonstrate a role for pRb during terminal differentiation of skeletal muscles in vivo and place pRb at a nodal point that controls cell proliferation, differentiation, and death.[Key Words: Retinoblastoma gene; differentiation; myogenesis; cell cycle control; apoptosis] Received July 25, 1996; revised version accepted October 16, 1996.Terminal differentiation is a dynamic process coupled to cell cycle arrest that requires continuous active control (Blau 1993). The retinoblastoma gene (RB1) product (pRB) has been implicated in cell cycle exit and terminal differentiation. For example, SV40 large T antigen, which binds and inactivates pRb, can stimulate differentiated myotubes in culture to reenter the cell cycle (Gu et al. 1993). RB1 is a tumor suppressor gene, absence of which predisposes individuals to retinoblastoma in infancy and, to a lesser extent, osteosarcoma in the second decade of life, at times when these tissues normally undergo terminal differentiation (for review, see Zacksenhaus et al. 1993a). Inactivation of RB1 also contributes to the malignant progression of a wide spectrum of tumors including breast, prostate, lung, and bladder. Moreover, tumors with apparently normal RB1 frequently contain mutations in the pathway that regulates pRb function, 4These authors contributed equally to this work. 5Corresponding authors. resulting in inactivation of pRb (for review, see Weinberg 1995).pRb is a member of a family of proteins including p107 (Ewen et al. 1991) and p130 (Harmon et al. 1993;Li et al. 1993) that interact with transcription factors and viral oncoproteins through shared conserved domains. The RB family of proteins exerts a negative effect on cell proliferation by modulating the activity of certain transcription factors (Defeo-Jones et al.

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“…It will be of interest to determine if such differences extend to p107 by assessing its relative capacity to inhibit DNA replication. Second, antagonists of Rb family members, such as SV40 large-T antigen, induce unscheduled DNA synthesis and aneuploidy in certain cell types (27,67,71). Whether the binding of viral oncoproteins to Rb-family members results in their dissociation from MCM7 is currently under investigation.…”

Section: Discussionmentioning

confidence: 99%

Negative Regulation of DNA Replication by the Retinoblastoma Protein Is Mediated by Its Association with MCM7

Sterner¹,

Dew-Knight²,

Musahl

et al. 1998

Molecular and Cellular Biology

183

170

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A yeast two-hybrid screen was employed to identify human proteins that specifically bind the amino-terminal 400 amino acids of the retinoblastoma (Rb) protein. Two independent cDNAs resulting from this screen were found to encode the carboxy-terminal 137 amino acids of MCM7, a member of a family of proteins that comprise replication licensing factor. Full-length Rb and MCM7 form protein complexes in vitro, and the amino termini of two Rb-related proteins, p107 and p130, also bind MCM7. Protein complexes between Rb and MCM7 were also detected in anti-Rb immunoprecipitates prepared from human cells. The amino-termini of Rb and p130 strongly inhibited DNA replication in an MCM7-dependent fashion in a Xenopus in vitro DNA replication assay system. These data provide the first evidence that Rb and Rb-related proteins can directly regulate DNA replication and that components of licensing factor are targets of the products of tumor suppressor genes.The retinoblastoma (Rb) susceptibility gene, Rb-1, is paradigmatic for a class of evolutionarily conserved genes variously termed tumor suppressor genes, anti-oncogenes, or recessive oncogenes. Deletion or mutational inactivation of Rb-1 is associated with the genesis of a variety of human cancers, including retinoblastoma, osteosarcoma, and small cell lung, bladder, and breast carcinomas (for a review, see reference 83). Mice hemizygous for Rb function are predisposed to a distinct spectrum of neoplasms, exhibiting an increased susceptibility to the development of brain, pituitary, and thyroid tumors (11,43,49). In addition to negatively regulating cell proliferation, Rb also functions to induce and/or maintain cell differentiation. For example, mice nullizygous for Rb function perish in utero and exhibit defects in the differentiation of hematopoietic, nervous, lens, and muscle tissues (11,43,49,56,61,73,88). Rb is also involved in a distinct pathway of deregulated cell growth and tumorigenesis, which is transformation induced by DNA tumor viruses. The E1A protein of adenovirus, large-T antigens of simian virus 40 (SV40) and polyomaviruses, and the E7 protein of human papillomaviruses all form physical complexes with the Rb protein, and such interactions abrogate Rb-mediated growth suppression (16,22,23,63,85). That each of these virus families has evolved independently to bind Rb underscores the idea that this tumor suppressor gene functions at one or more critical checkpoints in the regulation of cell growth.Rb-1 encodes a ubiquitously expressed set of nuclear proteins, termed p105-Rb, that are subject to cyclical waves of phosphorylation by cyclin-dependent kinases (7,8,51,55,60,80,83). Quiescent, terminally differentiated cells and cells in early portions of the cell cycle carry largely unphosphorylated p105-Rb. Shortly before the initiation of DNA synthesis, Rb is phosphorylated by cyclin D-and E-associated kinases and becomes increasingly modified as cells progress through S phase and G 2 (15,83). Rb is abruptly dephosphorylated at the end of mitosis probably by a ty...

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Alterations of DNA content in human endometrial stromal cells transfected with a temperature-sensitive SV40: tetraploidization and physiological consequences

Cited by 12 publications

References 0 publications

CRISPR/Cas9-Induced (CTG⋅CAG) n Repeat Instability in the Myotonic Dystrophy Type 1 Locus: Implications for Therapeutic Genome Editing

CRISPR/Cas9-Induced (CTG⋅CAG) n Repeat Instability in the Myotonic Dystrophy Type 1 Locus: Implications for Therapeutic Genome Editing

pRb controls proliferation, differentiation, and death of skeletal muscle cells and other lineages during embryogenesis.

Negative Regulation of DNA Replication by the Retinoblastoma Protein Is Mediated by Its Association with MCM7

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