Adenoma multiplicity in irradiated Apc(Min) mice is modified by chromosome 16 segments from BALB/c

Ionizing radiation (IR) is a well-characterized carcinogen in humans and mice. The BALB/c mouse strain is unusually sensitive to IR-induced tissue damage and cancer development in a range of organs, suggestive of a partial defect in DNA damage response. This has been confirmed by finding BALB/c-specific functional polymorphism in Prkdc, a gene on mouse chromosome 16 that encodes the catalytic subunit of DNA-dependent protein kinase. Prkdc(BALB) has been associated with increased susceptibility to IR-induced mammary and lymphatic neoplasia. Here, we provide evidence that chromosome 16 segments from BALB/c interact with Apc(Min) (multiple intestinal neoplasia) and specifically enhance IR-induced adenoma development in the upper part of the small intestine.     

BALB/c alleles for Prkdc and Cdkn2a interact to modify tumor susceptibility in Trp53+/- mice

In mice heterozygous for p53 (Trp53(+/-)), the incidence of mammary tumors varies among strains, with C57BL/6 being resistant and BALB/c being susceptible. Mammary tumor phenotypes were examined in female Trp53(+/-) F1 mice (C57BL/6 x BALB/c;n = 19) and N2 backcross mice [(C57BL/6 x BALB/c) x BALB/c] (n = 224). Susceptibility to mammary tumors segregated as a dominant phenotype in F1 females, but a higher frequency and shorter latency in N2 mice indicated a contribution from recessive-acting modifiers. Segregation of the hypomorphic BALB/c alleles for DNA-dependent protein kinase catalytic subunit (Prkdc) and p16(INK4A) (Cdkn2a) was analyzed in the N2 mice. The time to first tumor (considering all tumor types) was significantly different among the four genotype combinations (P = 0.01). Cdkn2a had a strong effect (P = 0.008) but was restricted to Prkdc(B/B) mice (P = 0.001), indicating a strong interaction between the loci. Differences in mammary tumor occurrence among genotypes for Prkdc and Cdkn2a in N2 mice were not statistically significant. This study indicates that BALB/c Prkdc and Cdkn2a alleles do modify tumor incidence in Trp53(+/-) mice and highlights the complexity of gene interaction effects in determining cancer phenotypes but discounts these alleles as major recessive loci contributing to spontaneous mammary tumor susceptibility.     

Radiation induces genomic instability and mammary ductal dysplasia in Atm heterozygous mice

Ataxia-telangiectasia (AT) is a genetic syndrome resulting from the inheritance of two defective copies of the ATM gene that includes among its stigmata radiosensitivity and cancer susceptibility. Epidemiological studies have demonstrated that although women with a single defective copy of ATM (AT heterozygotes) appear clinically normal, they may never the less have an increased relative risk of developing breast cancer. Whether they are at increased risk for radiation-induced breast cancer from medical exposures to ionizing radiation is unknown. We have used a murine model of AT to investigate the effect of a single defective Atm allele, the murine homologue of ATM, on the susceptibility of mammary epithelial cells to radiation-induced transformation. Here we report that mammary epithelial cells from irradiated mice with one copy of Atm truncated in the PI-3 kinase domain were susceptible to radiation-induced genomic instability and generated a 10% incidence of dysplastic mammary ducts when transplanted into syngenic recipients, whereas cells from Atm(+/+) mice were stable and formed only normal ducts. Since radiation-induced ductal dysplasia is a precursor to mammary cancer, the results indicate that AT heterozygosity increases susceptibility to radiogenic breast cancer in this murine model system.     

Variations in Prkdc encoding the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) and susceptibility to radiation-induced apoptosis and lymphomagenesis.

DNA double-strand breaks (DSBs) induced by ionizing radiation enforce cells to die, if unrepaired; while if misrepaired, DSBs may cause malignant transformation. The DSB repair system predominant in mammals requires DNA-dependent protein kinase (DNA-PK). Previously, we identified the apoptosis susceptibility gene Radiation-induced apoptosis 1 (Rapop1) on mouse chromosome 16. The STS/A (STS) allele at Rapop1 leads to decreased sensitivity to apoptosis in the BALB/cHeA (BALB/c) background. In the present study, we established Rapop1 congenic strains C.S-R1 and C.S-R1L, which contain the STS genome in a 0.45 cM interval critical for Rapop1 in common in the BALB/c background. Within the segment critical for Rapop1, Prkdc encoding the catalytic subunit of DNA-PK (DNA-PKcs) was assigned. Two variations T6,418C and G11,530A, which induce amino acid substitutions C2,140R downstream from the putative leucine zipper motif and V3,844M near the kinase domain, respectively, were found between BALB/c and STS for Prkdc. The majority of inbred strains such as C57BL/6J carried the STS allele at Prkdc; a few strains including 129/SvJ and C.B17 carried the BALB/c allele. DNA-PK activity as well as DNA-PKcs expression was profoundly diminished in BALB/c and 129/SvJ mice as compared with C57BL/6 and C.S-R1 mice. In the crosses (C.S-R1 x BALB/c)F(1) x 129/SvJ and (C.S-R1 x BALB/c)F(1) x C.B17, enhanced apoptosis occurred in the absence of the wild-type allele at Prkdc. C.S-R1 and C.S-R1L were both less sensitive to radiation lymphomagenesis than BALB/c. Our study provides strong evidence for Prkdc as a candidate for Rapop1 and a susceptibility gene for radiation lymphomagenesis as well.     

DNA protein kinase-dependent G2 checkpoint revealed following knockdown of ataxia-telangiectasia mutated in human mammary epithelial cells

Members of the phosphatidylinositol 3-kinase-related kinase family, in particular the ataxia-telangiectasia mutated (ATM) kinase and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), regulate cellular responses to DNA double-strand breaks. Increased sensitivity to ionizing radiation (IR) in DNA-PKcs- or ATM-deficient cells emphasizes their important roles in maintaining genome stability. Furthermore, combined knockout of both kinases is synthetically lethal, suggesting functional complementarity. In the current study, using human mammary epithelial cells with ATM levels stably knocked down by >90%, we observed an IR-induced G(2) checkpoint that was only slightly attenuated. In marked contrast, this G(2) checkpoint was significantly attenuated with either DNA-PK inhibitor treatment or RNA interference knockdown of DNA-PKcs, the catalytic subunit of DNA-PK, indicating that DNA-PK contributes to the G(2) checkpoint in these cells. Furthermore, in agreement with the checkpoint attenuation, DNA-PK inhibition in ATM-knockdown cells resulted in reduced signaling of the checkpoint kinase CHK1 as evidenced by reduced CHK1 phosphorylation. Taken together, these results show a DNA-PK-dependent component to the IR-induced G(2) checkpoint, in addition to the well-defined ATM-dependent component. This may have important implications for chemotherapeutic strategies for breast cancers.     

Synergistic radiosensitizing effect of BR101801, a specific DNA-dependent protein kinase inhibitor,in various human solid cancer cells and xenografts

DNA-dependent protein kinase (DNA-PK), an essential component of the non-homologous end-joining (NHEJ) repair pathway, plays an important role in DNA damage repair (DDR). Therefore, DNA-PK inhibition is a promising approach for overcoming radiotherapy or chemotherapy resistance in cancers. In this study, we demonstrated that BR101801, a potent DNA-PK inhibitor, acted as an effective radiosensitizer in various human solid cancer cells and an in vivo xenograft model. Overall, BR101801 strongly elevated ionizing radiation (IR)-induced genomic instability via induction of cell cycle G₂/M arrest, autophagic cell death, and impairment of DDR pathway in human solid cancer cells. Interestingly, BR101801 inhibited not only phosphorylation of DNA-PK catalytic subunit in NHEJ factors but also BRCA2 protein level in homologous recombination (HR) factors. In addition, combination BR101801 and IR suppressed tumor growth compared with IR alone by reducing phosphorylation of DNA-PK in human solid cancer xenografts. Our findings suggested that BR101801 is a selective DNA-PK inhibitor with a synergistic radiosensitizing effect in human solid cancers, providing evidence for clinical applications.     

Threonines 2638/2647 in DNA-PK are essential for cellular resistance to ionizing radiation

DNA-dependent protein kinase (DNA-PK) is required for the repair of double-stranded DNA breaks through the nonhomologous DNA end joining pathway. DNA-PK activity is required for DNA repair, but kinase activity also appears to be attenuated through an autoregulatory feedback loop. We show that autophosphorylation of DNA-PK catalytic subunit occurs in trans at least three sites NH(2) terminal to the catalytic domain and that two sites, threonine 2638 and 2647, determine DNA-PK autophosphorylation in vitro. Thr2638/2647ala substitution in DNA-PK catalytic subunit compromised cellular resistance to ionizing radiation without affecting DNA end joining, suggesting a requirement for DNA-PK inactivation for cell survival at a step after the rejoining of double-stranded DNA breaks.     

DNA修复基因BRCA1和DNA-PKcs在放射治疗中有很大的潜能

Radiotherapy is a part of the front-line treatment regime for many cancers. The mechanisms of radiation-induced effects in cancers mainly involves double-strand breaks (DBS) which plays very important role in maintaining the stability of gene. As DNA repair gene breast cancer 1 (BRCA1) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) can act to maintain genetic stability though two distinct and complementary mechanisms for DNA DSB repair-homologous recombination (HR) and non-homologous end joining (NHEJ). Therefor, BRCA1 and DNA-PKcs are closely associated with radiation sensitivity, which means that they may be used as a useful tool to predict radio sensitivity in human tumour cells.     

Role of DNA-dependent protein kinase catalytic subunit in cancer development and treatment

DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key component of the non-homologous end-joining (NHEJ) pathway, is involved in DNA double-strand break repair, immunocompetence, genomic integrity, and epidermal growth factor receptor signaling. Clinical studies indicate that expression and activity of DNA-PKcs is correlated with cancer progression and response to treatment. Various anti-DNA-PKcs strategies have been developed and tested in preclinical studies to exploit the benefit of DNA-PKcs inhibition in sensitization of radiotherapy and in combined modality therapy with other antitumor agents. In this article, we review the association between DNA-PKcs and cancer development and discuss current approaches and mechanisms for inhibition of DNA-PKcs. The future challenges are to understand how DNA-PKcs activity is correlated with cancer susceptibility and to identify those patients who would most benefit from DNA-PKcs inhibition.     

A polymorphism at codon 133 of the tumor suppressor RASSF1A is associated with tumorous alteration of the breast

The tumor suppressor gene RASSF1A is inactivated or mutated in different tumor entities including breast cancer. The frequency of the genomic variants of RASSF1A in patients with breast tumors has not been evaluated. We studied the association between ten nucleotide polymorphisms of RASSF1A and the risk of breast cancer in 178 cases with tumorous alterations of mammary tissue (including 141 carcinomas and 37 fibroadenomas) and 70 controls by SSCP and sequencing. Polymorphisms of RASSF1A were found at codon 28 and codon 133. The distribution of polymorphisms at codon 28 showed no significant difference between the patient groups: 5 of 178 (2.8%) in patients with tumorous alterations and 2 of 70 (2.9%) in control patients. However, the Gright curved arrow T polymorphism (GCTright curved arrow TCT; Alaright curved arrow Ser) at codon 133, which alters the microtubule association and stabilization domain of RASSF1A, exhibited a different genotype distribution: 29 out of 141 (20.6%) patients with breast carcinoma and 9 out of 37 (24.3%) patients with fibroadenoma harbored mutant T-alleles. However, only in 2 out of 70 (2.9%) controls, the mutant T-allele was detected and therefore the frequency was significantly diminished compared to tumorous alterations (Fisher's exact test: carcinomas vs. controls, p = 0.0003; fibroadenoma vs. controls, p = 0.001). From five probands with homozygous TT-genotype at codon 133, three were diagnosed with carcinomas and two with fibroadenomas. Our data indicate that the mutant T-allele of RASSF1A at codon 133 is correlated with an increased number of breast tumors.     

Mouse strain (STS/A) resistant to mammary tumor induction by hypophysial isografts

Implantation of hypophysial isografts does not lead to induction of mammary tumors in all strains of mice lacking the exogenous murine mammary tumor virus. While O20, C3Hf, and BALB/c females are highly susceptible and C57BL and TSI females are of intermediate susceptibility, the STS females appear to be nearly totally resistant. The resistance of the STS strain is not due to failure of prolactin production by the hypophysial isografts and may therefore be due to a genetically controlled mechanism at target cell level. Neither resistance, i.e., low incidence of mammary tumors (2% in STS), nor susceptibility, i.e., high incidence at low age (93% at 349 days in C3Hf; 83% at 360 days in BALB/c), is dominant. F1 hybrids of strain STS and the two strains C3Hf and BALB/c show high incidences (STS X C3Hf F1, 90%; STS X BALB/c F1, 60%), but the age at which tumors appear (476 and 604 days, respectively) is much higher, suggesting that more than one gene is involved in this type of hormonal carcinogenesis of the mammary gland in mice.     

ras gene mutations are absent in NMU-induced mammary carcinomas from aging rats

Carcinoma induction in the rat mammary carcinogenesis model is age dependent. In this study, mammary cancer susceptibility and ras gene activation were investigated in rats exposed to N:-methyl-N:-nitrosourea (NMU) at 2, 6, 8 and 15 months. Animals were resistant to NMU-induced mammary tumor development when exposed at 6 and 8 months of age, whereas a significant number of mammary carcinomas developed in animals exposed to NMU at 2 and 15 months of age. G35-->A35 activating mutations in the Harvey ras gene were found only in mammary carcinomas from rats exposed to NMU at 2 months of age, but not in tumors that developed in animals exposed to NMU at 15 months of age. No G35-->A35 activating mutations were present in the Kirsten ras gene of any of the mammary tumors. Additional analysis of exons 1 and 2 of the Harvey ras gene from mammary carcinomas that developed in animals exposed to NMU at 15 months of age did not reveal any other activating mutations in this gene. In mammary carcinomas from animals exposed to NMU at 2 months of age, the frequency of mammary carcinomas with mutations in the Harvey ras gene was independent of the time from which the tumor first appeared. Therefore, age at the time of carcinogen exposure plays a critical role in both breast cancer susceptibility and the molecular events that contribute to mammary carcinoma development.     

YB-1 evokes susceptibility to cancer through cytokinesis failure, mitotic dysfunction and HER2 amplification

Y-box binding protein-1 (YB-1) expression in the mammary gland promotes breast carcinoma that demonstrates a high degree of genomic instability. In the present study, we developed a model of pre-malignancy to characterize the role of this gene during breast cancer initiation and early progression. Antibody microarray technology was used to ascertain global changes in signal transduction following the conditional expression of YB-1 in human mammary epithelial cells (HMEC). Cell cycle-associated proteins were frequently altered with the most dramatic being LIM kinase 1/2 (LIMK1/2). Consequently, the misexpression of LIMK1/2 was associated with cytokinesis failure that acted as a precursor to centrosome amplification. Detailed investigation revealed that YB-1 localized to the centrosome in a phosphorylation-dependent manner, where it complexed with pericentrin and γ-tubulin. This was found to be essential in maintaining the structural integrity and microtubule nucleation capacity of the organelle. Prolonged exposure to YB-1 led to rampant acceleration toward tumorigenesis, with the majority of cells acquiring numerical and structural chromosomal abnormalities. Slippage through the G(1)/S checkpoint due to overexpression of cyclin E promoted continued proliferation of these genomically compromised cells. As malignancy further progressed, we identified a subset of cells harboring HER2 amplification. Our results recognize YB-1 as a cancer susceptibility gene, with the capacity to prime cells for tumorigenesis.     

Mutational analysis of the transforming growth factor beta receptor type II gene in hereditary nonpolyposis colorectal cancer and early-onset colorectal cancer patients.

Somatic mutations in the transforming growth factor beta receptor type II (TGF-beta RII) gene have been observed in various human cancers showing microsatellite instability. Most of the mutations observed were additions or deletions of the mononucleotide repeat sequence present in TGF-beta RII coding region, suggesting that the TGF-beta RII may be a target gene of genomic instability in tumorigenesis. Recently, we reported germ-line frameshift mutations in the mononucleotide repeat sequence of the hMSH6 gene, which is believed to be one of the target genes of genomic instability in tumorigenesis, suggesting the possibility of germ-line mutation in mononucleotide repeat sequences. Moreover, one case of germ-line mutation in the TGF-beta RII gene was identified in a hereditary nonpolyposis colorectal cancer (HNPCC) kindred, indicating the involvement of TGF-beta RII inactivation in tumorigenesis of HNPCC. However, germ-line mutation analysis of all of the coding sequences and the mononucleotide repeat sequence of the TGF-beta RII in HNPCC patients has not yet been fully elucidated. Therefore, to further investigate the presence of germ-line mutations, we screened all of the coding region sequences and mononucleotide repeat sequence of TGF-beta RII from 35 HNPCC, 44 suspected HNPCC, and 45 sporadic early-onset colorectal cancer patients. However, no pathogenic mutations other than silent mutations, introgenic mutation, and polymorphisms were identified. Two silent mutations at codons 309 (ACG to ACA) and 340 (CAT to CAC) in the kinase domain located in exon 4 were detected. A 1-bp cytidine deletion was observed 6 bases from the 3' end of intron. Two polymorphisms were identified at codon 389 (AAC to AAT) and at the fourth-to-last base in intron 3. The polymorphism at codon 389 was more frequent in HNPCC (20%; 7 of 35) and suspected HNPCC patients (18%; 8 of 44) than in nonmalignant control group (10%; 5 of 50). Moreover, the frequency was significantly higher in early-onset colorectal cancer patients (31%; 14 of 45). This is the first report of a different frequency of polymorphism in HNPCC, suspected HNPCC, early-onset colorectal cancer patients, and healthy normal individuals. This result suggests that: (a) germ-line mutation of the TGF-beta RII gene may be a rare event during tumorigenesis in HNPCC and sporadic early-onset colorectal cancer; (b) the mononucleotide repeat sequence of the TGF-beta RII gene is an apparent target of genomic instability but not of germ-line mutation; and (c) the polymorphism of codon 389 (AAC to AAT) is frequent, especially in early-onset colorectal cancer patients, in which it is more frequent than in control group.     

Is MSH2 a breast cancer susceptibility gene?

Mutations in the DNA mismatch repair gene MSH2 lead to increased replication error and microsatellite instability and account for a substantial proportion of hereditary non-polyposis colorectal cancer (Lynch syndrome). A recent international collaborative genome-wide linkage scan (GWS) for breast cancer susceptibility loci found some evidence for there being a breast cancer susceptibility gene in a genomic region on chromosome 2p close to MSH2. We sought to investigate the possibility that mutations in MSH2 might explain the multiple cases of breast cancer in some families that were included in the international GWS. DNA samples from the affected probands of 59 multiple-case breast cancer families, many of whom gave LOD scores >0.5 in the MSH2 region, were screened for large genomic alterations in MSH2 via the Multiplex Ligation-dependant Probe Amplification (MLPA) assay and for coding region mutations via exonic sequencing. Several of the families also contained cases of colorectal cancer in addition to breast cancer and had been included in the GWS that had identified a positive LOD score on chromosome 2p. Using MLPA, c.1236C > T was identified in one proband but this variant was not predicted to create an alternate acceptor/donor site within exon 7 MSH2 using in silico analyses. A c.1734T > C was identified in a second proband via exonic sequencing but testing of the variant in other family members did not support segregation of this variant with disease. Extensive screening of 59 multiple-case breast cancer families did not identify any coding region mutations or larger genomic alterations in MSH2 that might implicate MSH2 as a breast cancer susceptibility gene.     

The Catalytic Subunit of DNA-Dependent Protein Kinase Coordinates with Polo-Like Kinase 1 to Facilitate Mitotic Entry

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is the key regulator of the non-homologous end joining pathway of DNA double-strand break repair. We have previously reported that DNA-PKcs is required for maintaining chromosomal stability and mitosis progression. Our further investigations reveal that deficiency in DNA-PKcs activity caused a delay in mitotic entry due to dysregulation of cyclin-dependent kinase 1 (Cdk1), the key driving force for cell cycle progression through G₂/M transition. Timely activation of Cdk1 requires polo-like kinase 1 (Plk1), which affects modulators of Cdk1. We found that DNA-PKcs physically interacts with Plk1 and could facilitate Plk1 activation both in vitro and in vivo. Further, DNA-PKcs–deficient cells are highly sensitive to Plk1 inhibitor BI2536, suggesting that the coordination between DNA-PKcs and Plk1 is not only crucial to ensure normal cell cycle progression through G₂/M phases but also required for cellular resistance to mitotic stress. On the basis of the current study, it is predictable that combined inhibition of DNA-PKcs and Plk1 can be employed in cancer therapy strategy for synthetic lethality.Abbreviations: DNA-PKcs, DNA-dependent protein kinase catalytic subunit; Plk1, polo-like kinase 1; PBD, polo box domain