Influence of proliferation on DNA repair rates in liver

Clair, William H. St.; Dwarakanath, Bilikere S.; Zhang, Hong; Wheeler, Kenneth T.

doi:10.1016/0014-4827(91)90440-6

Cited by 10 publications

(1 citation statement)

References 10 publications

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“…Cellular quiescence has been associated with less active DNA repair pathways, which are a key component of successful site‐specific gene editing . To characterize the changes in hepatocyte gene expression in ex vivo cultured mouse hepatocytes , we performed a comparative qPCR on five genes required for homologous recombination: Brca1 , Brca2 , Rad51 , Rad52 , and Pcna .…”

Section: Resultsmentioning

confidence: 99%

Ex Vivo Hepatocyte Reprograming Promotes Homology‐Directed DNA Repair to Correct Metabolic Disease in Mice After Transplantation

VanLith¹,

Guthman²,

Nicolas³

et al. 2019

Hepatol Commun

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Ex vivo CRISPR/Cas9‐mediated gene editing in hepatocytes using homology‐directed repair (HDR) is a potential alternative curative therapy to organ transplantation for metabolic liver disease. However, a major limitation of this approach in quiescent adult primary hepatocytes is that nonhomologous end‐joining is the predominant DNA repair pathway for double‐strand breaks (DSBs). This study explored the hypothesis that ex vivo hepatocyte culture could reprogram hepatocytes to favor HDR after CRISPR/Cas9‐mediated DNA DSBs. Quantitative PCR (qPCR), RNA sequencing, and flow cytometry demonstrated that within 24 hours, primary mouse hepatocytes in ex vivo monolayer culture decreased metabolic functions and increased expression of genes related to mitosis progression and HDR. Despite the down‐regulation of hepatocyte function genes, hepatocytes cultured for up to 72 hours could robustly engraft in vivo . To assess functionality long‐term, primary hepatocytes from a mouse model of hereditary tyrosinemia type 1 bearing a single‐point mutation were transduced ex vivo with two adeno‐associated viral vectors to deliver the Cas9 nuclease, target guide RNAs, and a 1.2‐kb homology template. Adeno‐associated viral Cas9 induced robust cutting at the target locus, and, after delivery of the repair template, precise correction of the point mutation occurred by HDR. Edited hepatocytes were transplanted into recipient fumarylacetoacetate hydrolase knockout mice, resulting in engraftment, robust proliferation, and prevention of liver failure. Weight gain and biochemical assessment revealed normalization of metabolic function. Conclusion: The results of this study demonstrate the potential therapeutic effect of ex vivo hepatocyte‐directed gene editing after reprogramming to cure metabolic disease in a preclinical model of hereditary tyrosinemia type 1.

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Section: Resultsmentioning

confidence: 99%

Ex Vivo Hepatocyte Reprograming Promotes Homology‐Directed DNA Repair to Correct Metabolic Disease in Mice After Transplantation

VanLith¹,

Guthman²,

Nicolas³

et al. 2019

Hepatol Commun

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Contribution of O⁶‐methylguanine‐DNA methyltransferase to monofunctional alkylating‐agent resistance in human brain tumor—derived cell lines

Bobola

Blank

Berger

et al. 1995

Molecular Carcinogenesis

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The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) has been implicated in resistance of human brain tumors to alkylating agents. We observed that 14 human medulloblastoma- and glioma-derived cell lines differ in sensitivity to the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), as shown by their 28-fold range in 10% survival dose (LD10). By using the substrate analogue inhibitor O6-benzylguanine (O6-BG), we showed that the contribution of MGMT to resistance varies widely, as evidenced by 3- to 30-fold reductions in LD10 among the lines, and varies up to 20-fold among subpopulations of individual lines. Importantly, variability in resistance, manifested as a 20-fold range in LD10, persists after measurable MGMT is eliminated, disclosing differential contributions of other resistance mechanisms to survival. Cells exposed to MNNG while suspended in growth medium are more resistant than cells alkylated as subconfluent monolayers, and MGMT accounts for a smaller proportion of their resistance. Notably, the MGMT content of the lines is not statistically correlated with MNNG resistance or with potentiation of killing by O6-BG, even though MGMT is a biochemically demonstrated determinant of resistance. In contrast, the same lines vary less in resistance to the ethylating agent N-ethylnitrosourea (ENU), and MGMT makes only a small contribution to resistance. Our results strongly indicate that resistance to both MNNG and ENU is multifactorial.

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Contribution of O⁶‐methylguanine‐DNA methyltransferase to resistance to 1,3‐(2‐chloroethyl)‐1‐nitrosourea in human brain tumor‐derived cell lines

Bobola

Berger

Silber

1995

Molecular Carcinogenesis

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To assess the possible role of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) in resistance of brain neoplasms to the clinically important chloroethylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), we quantitated MGMT activity, BCNU survival, and the effect of ablating MGMT activity on the sensitivity of 14 human medulloblastoma- and glioma-derived cell lines. BCNU resistance, measured as 10% survival dose (LD10), differed eightfold among the lines. Elimination of measurable MGMT activity with the substrate analogue inhibitor O6-benzylguanine (O6-BG) revealed a variable but limited contribution of MGMT to survival. In no case did O6-BG reduce LD10 by more than 3.4-fold. In contrast, O6-BG reduced the LD10 for N-methyl-N'-nitro-N-nitrosoguanidine up to 31-fold in the same cell lines (Bobola MS, Blank A, Berger MS, Silber JR, Mol Carcinog 13:70-80, 1995). Variability in BCNU survival, manifested as a sevenfold range of LD10, persists after measurable MGMT was eliminated, indicating that another mechanism or mechanisms is operating to limit cytotoxicity. Cells alkylated while suspended in growth medium are more resistant to BCNU and display less dependence on MGMT than cells treated while proliferating on a plastic substratum. When alkylated in suspension, most of the lines are either unresponsive to O6-BG or contain a subpopulation that did not respond to O6-BG. Our results demonstrate that BCNU resistance is multifactorial and that MGMT makes a modest contribution to resistance in our lines.

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Influence of proliferation on DNA repair rates in liver

Cited by 10 publications

References 10 publications

Ex Vivo Hepatocyte Reprograming Promotes Homology‐Directed DNA Repair to Correct Metabolic Disease in Mice After Transplantation

Ex Vivo Hepatocyte Reprograming Promotes Homology‐Directed DNA Repair to Correct Metabolic Disease in Mice After Transplantation

Contribution of O⁶‐methylguanine‐DNA methyltransferase to monofunctional alkylating‐agent resistance in human brain tumor—derived cell lines

Contribution of O⁶‐methylguanine‐DNA methyltransferase to resistance to 1,3‐(2‐chloroethyl)‐1‐nitrosourea in human brain tumor‐derived cell lines

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Influence of proliferation on DNA repair rates in liver

Cited by 10 publications

References 10 publications

Ex Vivo Hepatocyte Reprograming Promotes Homology‐Directed DNA Repair to Correct Metabolic Disease in Mice After Transplantation

Ex Vivo Hepatocyte Reprograming Promotes Homology‐Directed DNA Repair to Correct Metabolic Disease in Mice After Transplantation

Contribution of O6‐methylguanine‐DNA methyltransferase to monofunctional alkylating‐agent resistance in human brain tumor—derived cell lines

Contribution of O6‐methylguanine‐DNA methyltransferase to resistance to 1,3‐(2‐chloroethyl)‐1‐nitrosourea in human brain tumor‐derived cell lines

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Contribution of O⁶‐methylguanine‐DNA methyltransferase to monofunctional alkylating‐agent resistance in human brain tumor—derived cell lines

Contribution of O⁶‐methylguanine‐DNA methyltransferase to resistance to 1,3‐(2‐chloroethyl)‐1‐nitrosourea in human brain tumor‐derived cell lines