In vitro rejoining of double strand breaks induced in cellular DNA by bleomycin and restriction endonucleases

SummaryIonising irradiation acts primarily via induction of DNA damage, among which doublestrand breaks are the most important lesions. These lesions may lead to lethal chromosome aberrations, which are the main reason for cell inactivation. Double-strand breaks can be repaired by several different mechanisms. The regulation of these mechanisms appears be fairly different for normal and tumour cells. Among different cell lines capacity of doublestrand break repair varies by only few percents and is known to be determined mostly by genetic factors. Knowledge about doublestrand break repair mechanisms and their regulation is important for the optimal application of ionising irradiation in medicine.

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Focused ultrasound radiosensitizes human cancer cells by enhancement of DNA damage

Strahlentherapie und Onkologie ◽

10.1007/s00066-021-01774-5 ◽

2021 ◽

Author(s):

Xinrui Zhang ◽

Mariana Bobeica ◽

Michael Unger ◽

Anastasia Bednarz ◽

Bjoern Gerold ◽

...

Keyword(s):

Prostate Cancer ◽

Dna Damage ◽

Cancer Cells ◽

Metabolic Activity ◽

Focused Ultrasound ◽

Double Strand Breaks ◽

High Intensity Focused Ultrasound ◽

Dna Double Strand Breaks ◽

Strand Breaks

Abstract Purpose High-intensity focused ultrasound (HIFU/FUS) has expanded as a noninvasive quantifiable option for hyperthermia (HT). HT in a temperature range of 40–47 °C (thermal dose CEM43 ≥ 25) could work as a sensitizer to radiation therapy (RT). Here, we attempted to understand the tumor radiosensitization effect at the cellular level after a combination treatment of FUS+RT. Methods An in vitro FUS system was developed to induce HT at frequencies of 1.147 and 1.467 MHz. Human head and neck cancer (FaDU), glioblastoma (T98G), and prostate cancer (PC-3) cells were exposed to FUS in ultrasound-penetrable 96-well plates followed by single-dose X‑ray irradiation (10 Gy). Radiosensitizing effects of FUS were investigated by cell metabolic activity (WST‑1 assay), apoptosis (annexin V assay, sub-G1 assay), cell cycle phases (propidium iodide staining), and DNA double-strand breaks (γH2A.X assay). Results The FUS intensities of 213 (1.147 MHz) and 225 W/cm2 (1.467 MHz) induced HT for 30 min at mean temperatures of 45.20 ± 2.29 °C (CEM43 = 436 ± 88) and 45.59 ± 1.65 °C (CEM43 = 447 ± 79), respectively. FUS improves the effect of RT significantly by reducing metabolic activity in T98G cells 48 h (RT: 96.47 ± 8.29%; FUS+RT: 79.38 ± 14.93%; p = 0.012) and in PC-3 cells 72 h (54.20 ± 10.85%; 41.01 ± 11.17%; p = 0.016) after therapy, but not in FaDu cells. Mechanistically, FUS+RT leads to increased apoptosis and enhancement of DNA double-strand breaks compared to RT alone in T98G and PC-3 cells. Conclusion Our in vitro findings demonstrate that FUS has good potential to sensitize glioblastoma and prostate cancer cells to RT by mainly enhancing DNA damage.

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No change in DNA damage or repair of single- and double-strand breaks as human diploid fibroblasts age in vitro

Experimental Cell Research ◽

10.1016/0014-4827(86)90494-5 ◽

1986 ◽

Vol 166 (2) ◽

pp. 497-509 ◽

Cited By ~ 19

Author(s):

Peter J. Mayer ◽

Matthews O. Bradley ◽

Warren W. Nichols

Keyword(s):

Dna Damage ◽

Double Strand Breaks ◽

Strand Breaks ◽

Diploid Fibroblasts ◽

Human Diploid Fibroblasts

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Homologous recombination and the repair of double-strand breaks during cotransformation of Dictyostelium discoideum

Molecular and Cellular Biology ◽

10.1128/mcb.8.7.2779-2786.1988 ◽

1988 ◽

Vol 8 (7) ◽

pp. 2779-2786

Author(s):

K S Katz ◽

D I Ratner

Keyword(s):

Homologous Recombination ◽

Dictyostelium Discoideum ◽

Strand Break ◽

Double Strand Breaks ◽

Restriction Endonucleases ◽

Linear Molecule ◽

Strand Breaks ◽

Transformation Vector ◽

Closed Circle ◽

Primary Vector

We examined the ability of unlinked nonreplicating plasmid molecules to undergo homologous recombination during cotransformation of Dictyostelium amoebae. The transformation vector B10S confers resistance to the antibiotic G418 and was always presented to amoebae as a closed circle. Cotransforming DNA, containing a slime mold cDNA and sequences homologous to the primary vector, was presented either as a closed circle or as a linear molecule after digestion with restriction endonucleases which cut within one of three distinct regions of the plasmid. Remarkably, homologous recombination occurred in every clone examined. Moreover, the products of recombination were identical in all instances, irrespective of the presence or position of linearized ends. The ends of the linear templates were not recombinogenic. Repair of the introduced double-strand break occurred frequently during recombination. The repair could occur intermolecularly or, more likely, intramolecularly, i.e., by recircularization. Many of the recombination events were of a nonreciprocal nature. Despite the startlingly frequent level of homologous recombination, the use of cotransforming DNA which contains no homology to the selected vector established that such recombination was not required for cotransformation.

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In Vitro Evaluation of No-carrier-added Radiolabeled Cisplatin ([189, 191Pt]cisplatin) Emitting Auger Electrons

10.20944/preprints202103.0621.v1 ◽

2021 ◽

Author(s):

Honoka Obata ◽

Atsushi B. Tsuji ◽

Hitomi Sudo ◽

Aya Sugyo ◽

Katsuyuki Minegishi ◽

...

Keyword(s):

Gel Electrophoresis ◽

Short Range ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Primary Target ◽

Auger Electrons ◽

Strand Breaks ◽

Chemical Damage ◽

No Carrier Added

Due to their short range (2–500 nm), Auger electrons (Auger e-) have the potential to induce nano-scale physiochemical damage to biomolecules. Although DNA is the primary target of Au-ger e-, it remains challenging to maximize the interaction between Auger e- and DNA. To assess the DNA-damaging effect of Auger e- released as close as possible to DNA without chemical damage, we radio-synthesized no-carrier-added (n.c.a.) [189, 191Pt]cisplatin and evaluated both its in vitro properties and DNA-damaging effect. Cellular uptake, intracellular distribution, and DNA binding were investigated, and DNA double-strand breaks (DSBs) were evaluated by im-munofluorescence staining of γH2AX and gel electrophoresis of plasmid DNA. Approximately 20% of intracellular radio-Pt was in a nucleus, and about 2% of intra-nucleus radio-Pt bound to DNA, although uptake of n.c.a. radio-cisplatin was low (0.6% incubated dose after 25-h incuba-tion), resulting in the frequency of cells with γH2AX foci was low (1%). Nevertheless, some cells treated with radio-cisplatin had γH2AX aggregates unlike non-radioactive cisplatin. These findings suggest n.c.a. radio-cisplatin binding to DNA causes severe DSBs by release of Auger e- very close to DNA without chemical damage by carriers. Efficient radio-drug delivery to DNA is necessary for successful clinical application of Auger e-.

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Effect of double-strand breaks on homologous recombination in mammalian cells and extracts

Molecular and Cellular Biology ◽

10.1128/mcb.5.12.3331-3336.1985 ◽

1985 ◽

Vol 5 (12) ◽

pp. 3331-3336

Author(s):

K Y Song ◽

L Chekuri ◽

S Rauth ◽

S Ehrlich ◽

R Kucherlapati

Keyword(s):

Homologous Recombination ◽

Mammalian Cells ◽

Recombination Frequency ◽

Double Strand Breaks ◽

Base Pairs ◽

In Vitro System ◽

Strand Breaks ◽

Cell Extracts ◽

Nuclear Extracts

We examined the effect of double-strand breaks on homologous recombination between two plasmids in human cells and in nuclear extracts prepared from human and rodent cells. Two pSV2neo plasmids containing nonreverting, nonoverlapping deletions were cotransfected into cells or incubated with cell extracts. Generation of intact neo genes was monitored by the ability of the DNA to confer G418r to cells or Neor to bacteria. We show that double-strand breaks at the sites of the deletions enhanced recombination frequency, whereas breaks outside the neo gene had no effect. Examination of the plasmids obtained from experiments involving the cell extracts revealed that gene conversion events play an important role in the generation of plasmids containing intact neo genes. Studies with plasmids carrying multiple polymorphic genetic markers revealed that markers located within 1,000 base pairs could be readily coconverted. The frequency of coconversion decreased with increasing distance between the markers. The plasmids we constructed along with the in vitro system should permit a detailed analysis of homologous recombinational events mediated by mammalian enzymes.

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Protective Activity of C-Geranylflavonoid Analogs from Paulownia tomentosa against DNA Damage in 137Cs Irradiated AHH-1Cells

Natural Product Communications ◽

10.1177/1934578x1400900919 ◽

2014 ◽

Vol 9 (9) ◽

pp. 1934578X1400900

Author(s):

Hyung-In Moon ◽

Min Ho Jeong ◽

Wol Soon Jo

Keyword(s):

Dna Damage ◽

Protective Effects ◽

Strand Breaks ◽

Human Lymphoblastoid Cell ◽

Wide Range ◽

Radiation Induced ◽

Anti Oxidant ◽

Dna Strand ◽

Cellular Dna

Radiotherapy is an important form of treatment for a wide range of cancers, but it can damage DNA and cause adverse effects. We investigated if the diplacone analogs of P. tomentosa were radio-protective in a human lymphoblastoid cell line (AHH-1). Four geranylated flavonoids, diplacone, 3′- O-methyl-5′-hydroxydiplacone, 3′- O-methyl-5′- O-methyldiplacone and 3′- O-methyldiplacol, were tested for their antioxidant and radio-protective effects. Diplacone analogs effectively scavenged free radicals and inhibited radiation-induced DNA strand breaks in vitro. They significantly decreased levels of reactive oxygen species and cellular DNA damage in 2 Gy-irradiated AHH-1 cells. Glutathione levels and superoxide dismutase activity in irradiated AHH-1 cells increased significantly after treatment with these analogs. The enhanced biological anti-oxidant activity and radioprotective activity of diplacone analogs maintained the survival of irradiated AHH-1 cells in a clonogenic assay. These data suggest that diplacone analogs may protect healthy tissue surrounding tumor cells during radiotherapy to ensure better control of radiotherapy and allow higher doses of radiotherapy to be employed.

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