DNA mismatch repair (MMR) processes the chemically-induced mispairs following treatment with clinically important nucleoside analogs such as 6-thioguanine (6-TG) and 5-fluorouracil (5-FU). MMR processing of these drugs has been implicated in activation of a prolonged G2/M cell cycle arrest for repair and later induction of apoptosis and/or autophagy for irreparable DNA damage. In this study, we investigated the role of BNIP3 in the activation of autophagy and the temporal relationship between a G2/M cell cycle arrest and the activation of BNIP3-mediated autophagy following MMR processing of 6-TG and 5-FU. We found that BNIP3 protein levels are up-regulated in a MLH1 (MMR+)-dependent manner following 6-TG and 5-FU treatment. Subsequent siRNA-mediated BNIP3 knockdown abrogates 6-TG -induced autophagy. We also found that p53 knockdown or inhibition of mTOR activity by rapamycin cotreatment impairs 6-TG and 5-FU-induced up-regulation of BNIP3 protein levels and autophagy. Furthermore, suppression of Chk1 expression and a subsequent reduction in 6-TG-induced G2/M cell cycle arrest by Chk1 siRNA promotes the extent of 6-TG-induced autophagy. These findings suggest that BNIP3 mediates 6-TG- and 5-FU induced autophagy in a p53- and mTOR-dependent manner. Additionally, the duration of Chk1-activated G2/M cell cycle arrest determines the level of autophagy following MMR processing of these nucleoside analogs.
BNIP3; p53; mTOR; autophagy; nucleoside analogs
Ribonucleotide reductase (RR), the rate limiting enzyme in the synthesis and repair of DNA, has been studied as a target for inhibition in the treatment of cancer for many years. While some researchers have focused on RR inhibitors as chemotherapeutic agents, particularly in hematologic malignancies, some of the most promising data has been generated in the field of radiosensitization. Early pre-clinical studies demonstrated that the addition of the first of these drugs, hydroxyurea, to ionizing radiation (IR) produced a synergistic effect in vitro, leading to a large number of clinical studies in the 1970–1980s. These studies, mainly in cervical cancer, initially produced a great deal of interest, leading to the incorporation of hydroxyurea in the treatment protocols of many institutions. However, over time, the conclusions from these studies have been called into question and hydroxyurea has been replaced in the standard of care of cervical cancer. Over the last 10 years, a number of well-done pre-clinical studies have greatly advanced our understanding of RR as a target. Those advances include the elucidation of the role of p53R2 and our understanding of the temporal relationship between the delivery of IR and the response of RR. At the same time, new inhibitors with increased potency and improved binding characteristics have been discovered, and pre-clinical and early clinical data look promising. Here we present a comprehensive review of the pre-clinical and clinical data in the field to date and provide some discussion of future areas of research.
ribonucleotide reductase; hydroxyurea; triapine; radiosensitizer; ionizing radiation; cervical cancer
A more flexible and higher-yielding in vitro DNA mismatch repair (MMR) substrate construction method, which was developed initially by Wang and Hays, is described for the construction of a nucleotide-based chemical mismatch (G/IU) and a G/T mismatch. Our modifications use the combination of two endonuclease enzymes (NheI and BciVI) and two new redesigned plasmids (pWDAH1A and pWDSH1B). In our modified methodology, plasmids are initially digested with the nicking endonucleases, followed by the streptavidin treatment. The mismatch-containing oligo is then annealed to the gap DNA and finally ligated to produce a mismatch-containing DNA substrate. We report a high efficiency (up to 90%) of these mismatch substrates and confirm recognition using a functional assay. These modifications, coupled with the use of the redesigned plasmids, can be applied for the construction of other types of chemically induced mismatches as well as insertion-deletion loops for future in vitro studies of MMR processing by our group and others.
mismatch repair; mismatch substrates construction; iododeoxyuridine
Purpose: Pre-operative chemoradiation (CRT) is currently the standard of care for patients with clinical stage II and III rectal cancer but only about 45% of patients achieve tumor downstaging and <20% of patients achieve a pathologic complete response. Better methods to stratify patients according to potential neoadjuvant treatment response are needed. We used microarray analysis to identify a genetic signature that correlates with a pathological complete response (pCR) to neoadjuvant CRT. We performed a gene network analysis to identify potential signaling pathways involved in determining response to neoadjuvant treatment.
Patients and Methods: We identified 31 T3–4 N0–1 rectal cancer patients who were treated with neoadjuvant fluorouracil-based CRT. Eight patients were identified to have achieved a pCR to treatment while 23 patients did not. mRNA expression was analyzed using cDNA microarrays. The correlation between mRNA expression and pCR from pre-treatment tumor biopsies was determined. Gene network analysis was performed for the genes represented by the predictive signature.
Results: A genetic signature represented by expression levels of the three genes EHBP1, STAT1, and GAPDH was found to correlate with a pCR to neoadjuvant treatment. The difference in expression levels between patients who achieved a pCR and those who did not was greatest for EHBP1. Gene network analysis showed that the three genes can be connected by the gene ubiquitin C (UBC).
Conclusion: This study identifies a 3-gene signature expressed in pre-treatment tumor biopsies that correlates with a pCR to neoadjuvant CRT in patients with clinical stage II and III rectal cancer. These three genes can be connected by the gene UBC, suggesting that ubiquitination is a molecular mechanism involved in determining response to treatment. Validating this genetic signature in a larger number of patients is proposed.
rectal neoplasms; ubiquitination; gene array; UBC; EHBP1
MLH1 is a key DNA mismatch repair (MMR) protein involved in maintaining genomic stability by participating in the repair of endogenous and exogenous mispairs in the daughter strands during S-phase. Exogenous mispairs can result following treatment with several classes of chemotherapeutic drugs as well as with ionizing radiation (IR). In this study, we investigated the role of the MLH1 protein in determining the cellular and molecular responses to prolonged low dose rate (LDR) IR, which is similar to the clinical use of cancer brachytherapy.
An isogenic pair of MMR+ (MLH1+) and MMR− (MLH1−) human colorectal cancer HCT116 cells were exposed to prolonged LDR-IR (1.3–17cGy/h × 24–96 h). The clonogenic survival and gene mutation rates were examined. Cell cycle distribution was analyzed with flow cytometry. Changes in selected DNA damage repair proteins, DNA damage response proteins and cell death marker proteins were examined with Western blotting.
MLH1+ HCT116 cells showed greater radiosensitivity with enhanced expression of apoptotic and autophagic markers; a reduced HPRT gene mutation rate; and more pronounced cell cycle alterations (increased late S population and a G2/M arrest) following LDR-IR compared to MLH1− HCT116 cells. Importantly, a progressive increase in MLH1 protein levels was found in MLH1+ cells during prolonged LDR-IR, which was temporally correlated with a progressive decrease in Rad51 protein (involved in homologous recombination, HR) levels.
MLH1 status significantly affects cellular responses to prolonged LDR-IR. MLH1 may enhance cell radiosensitivity to prolonged LDR-IR through inhibition of HR (via inhibition of Rad51).
mismatch repair; low dose rate IR; MLH1; Rad51; late S phase
For repair of damaged DNA, cells increase de novo synthesis of deoxyribonucleotide triphosphates through the rate-limiting, p53-regulated ribonucleotide reductase (RNR) enzyme. In this study we investigated whether pharmacological inhibition of RNR by 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, NSC #663249) enhanced chemoradiation sensitivity through a mechanism involving sustained DNA damage. RNR inactivation by 3-AP and resulting chemoradiosensitization were evaluated in human cervical (CaSki, C33-a) cancer cells through study of DNA damage (γ-H2AX signal) by flow cytometry, RNR subunit p53R2 and p21 protein steady-state levels by Western blot analysis and laser scanning imaging cytometry, and cell survival by colony formation assays. 3-AP treatment led to sustained radiation- and cisplatin-induced DNA damage (i.e. increased γ-H2AX signal) in both cell lines through a mechanism of inhibited RNR activity. Radiation, cisplatin and 3-AP exposure resulted in significantly elevated numbers and persistence of γ-H2AX foci that were associated with reduced clonogenic survival. DNA damage was associated with a rise in p53R2 but not p21 protein levels 6 h after treatment with radiation and/or cisplatin plus 3-AP. We conclude that blockage of RNR activity by 3-AP impairs DNA damage responses that rely on deoxyribonucleotide production and thereby may substantially increase chemoradiosensitivity of human cervical cancer.
The base excision repair (BER) pathway is required to repair endogenous and exogenous oxidative DNA damage. Multiple DNA repair pathways have been shown to be down-regulated in the tumor microenvironment, whereas APE1/Ref1, a central protein in BER, is overexpressed in many types of solid tumors. APE1/Ref1 has dual functions, participating both in BER and in redox regulation of oxidized transcription factors. Here, we show that inhibition of the BER pathway in an acidic tumor microenvironment increases oxidative DNA damage temporally related to increased intracellular reactive oxygen species. Unrepaired oxidative DNA damage results in cell cycle arrests and increased DNA double strand breaks, leading to cell death. Therefore, up-regulation of BER in solid cancers may represent an adaptive survival response. Consequently, BER inhibition may confer tumor microenvironment targeted cytotoxicity in human cancers. Our data suggest that BER inhibition is a rational basis for cancer therapy with or without other cytotoxic therapy. Additionally, our results offer insight as to why APE1/Ref1 retains it’s unique dual functionality, both of which counteract environmental oxidative stress.
base excision repair; APE1/Ref1; XRCC1; tumor microenvironment; DNA damage
5-iodo-2-pyrimidinone-2′-deoxyribose (IPdR) is a novel orally administered (po) prodrug of 5-iododeoxyuridine (IUdR). As po IPdR is being considered for clinical testing as a radiosensitizer in patients with high grade gliomas, we performed this in vivo study of IPdR-mediated cytotoxicity and radiosensitization in a human glioblastoma xenograft model, U87.
Methods and Materials
Groups of 8–9 athymic male nude mice (6–8 weeks old) were implanted with sc U87 xenograft tumors (4 × 106 cells) and then randomized to 10 treatment groups receiving increasing doses of po IPdR (0, 100, 250, 500, and 1000 mg/kg/d) administered once daily (qd) × 14 d with or without radiation therapy (RT) (0 or 2 Gy/d × 4 d) on days 11–14 of IPdR treatment. Systemic toxicity was determined by body weight measurements during and following IPdR treatment. Tumor response was assessed by changes in tumor volumes.
IPdR alone at doses of ≥500 mg/kg/d results in moderate inhibition of tumor growth. The combination of IPdR + RT results in a significant IPdR dose-dependent tumor growth delay with the maximum radiosensitization using ≥500 mg/kg/d. IPdR doses of 500 and 1000 mg/kg/d did result in transient 5–15% body weight loss during treatment.
In U87 human glioblastoma sc xenografts, po IPdR given qd × 14 d and RT given 2 Gy/d × 4 d (days 11–14 of IPdR treatment) results in a significant tumor growth delay in an IPdR dose-dependent pattern. The use of po IPdR + RT holds promise for phase I/II testing in patients with high grade gliomas.
IpdR; U87 xenografts; radiosensitization
This study assessed the safety/tolerability, pharmacokinetics, and clinical activity of three-times weekly intravenous 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, NSC #663249) in combination with once weekly intravenous cisplatin and daily pelvic radiation in patients with gynecologic malignancies. 3-AP is a novel small molecule inhibitor of ribonucleotide reductase (RNR) and is being tested as a potential radiosensitizer and chemosensitizer.
Patients with stage IB2-IVB cervical cancer (n=10) or recurrent uterine sarcoma (n=1) were assigned to dose-finding cohorts of 2-hour 3-AP infusions during five weeks of cisplatin chemoradiation. Pharmacokinetic and methemoglobin samples and tumor biopsy for RNR activity were obtained on days 1 and 10. Clinical response was assessed.
The maximum tolerated 3-AP dose is 25mg/m2 given three-times weekly during cisplatin and pelvic radiation. Two patients experienced manageable 3-AP-related grade 3 or 4 electrolyte abnormalities. 3-AP pharmacokinetics showed a 2-hour half-life, with median peak plasma concentrations of 277ng/mL (25mg/m2) and 467ng/mL (50mg/m2). Median methemoglobin levels peaked at 1% (25mg/m2) and 6% (50mg/m2) at 4 hours after initiating 3-AP infusions. No change in RNR activity was found on day 1 versus 10 in six early complete responders, while elevated RNR activity was seen on day 10 as compared to day 1 in four late complete responders (P =0.02). Ten (100%) patients with stage IB2-IVB cervical cancer achieved complete clinical response and remain without disease relapse with a median 18 months of follow-up (6-32 months).
3-AP was well tolerated at a three-times weekly intravenous 25mg/m2 dose during cisplatin and pelvic radiation.
Triapine; cervical cancer; ribonucleotide reductase; radiosensitization
Over the last 7 years, we have focused our experimental and computational research efforts on improving our understanding of the biochemical, molecular, and cellular processing of iododeoxyuridine (IUdR) and ionizing radiation (IR) induced DNA base damage by DNA mismatch repair (MMR). These coordinated research efforts, sponsored by the National Cancer Institute Integrative Cancer Biology Program (ICBP), brought together system scientists with expertise in engineering, mathematics, and complex systems theory and translational cancer researchers with expertise in radiation biology. Our overall goal was to begin to develop computational models of IUdR- and/or IR-induced base damage processing by MMR that may provide new clinical strategies to optimize IUdR-mediated radiosensitization in MMR deficient (MMR−) “damage tolerant” human cancers. Using multiple scales of experimental testing, ranging from purified protein systems to in vitro (cellular) and to in vivo (human tumor xenografts in athymic mice) models, we have begun to integrate and interpolate these experimental data with hybrid stochastic biochemical models of MMR damage processing and probabilistic cell cycle regulation models through a systems biology approach. In this article, we highlight the results and current status of our integration of radiation biology approaches and computational modeling to enhance IUdR-mediated radiosensitization in MMR− damage tolerant cancers.
mismatch repair; ionizing radiation; iododeoxyuridine; systems biology
Therapeutic ionizing radiation damages DNA, increasing p53-regulated ribonucleotide reductase (RNR) activity required for de novo synthesis of the deoxyribonucleotide triphosphates used during DNA repair. This study investigated the pharmacological inhibition of RNR in cells of virally or mutationally silenced p53 cancer cell lines using 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, Triapine® NSC #663249), a chemotherapeutic radiosensitizer that equally inhibits RNR M2 and p53R2 small subunits. The effects of 3-AP on RNR inhibition and resulting radiosensitization were evaluated in cervical (CaSki, HeLa and C33-a) and colon (RKO, RKO-E6) cancer cells. 3-AP treatment significantly enhanced radiation-related cytotoxicity in cervical and colon cancer cells. 3-AP treatment significantly decreased RNR activity, caused prolonged radiation-induced DNA damage, and resulted in an extended G1/S-phase cell cycle arrest in all cell lines. Similar effects were observed in both RKO and RKO-E6 cells, suggesting a p53-independent mechanism of radiosensitization. We conclude that inhibition of ribonucleotide reductase by 3-AP enhances radiation-mediated cytotoxicity independent of p53 regulation by impairing repair processes that rely on deoxyribonucleotide production, thereby substantially increasing the radiation sensitivity of human cancers.
Purpose. Typical treatment of retroperitoneal sarcomas (RPSs) is surgery with or without radiation therapy for localized disease. With surgery alone, local failure rates are as high as 90%; this led to radiation therapy playing an important role in the treatment of RPSs. Methods. Thirty-one patients with retroperitoneal sarcoma treated with gross total resection and radiation therapy make up this retrospective analysis. Nineteen were treated preoperatively and 12 postoperatively (median dose, 59.4 Gy)—sixteen also received intraoperative radiation therapy (IORT) (median dose, 11 Gy). Patients were followed with stringent regimens, including frequent CT scans of the chest, abdomen, and pelvis. Results. With a median follow-up of 19 months (range 1–66 months), the 2-year overall survival (OS) rate is 70% (median, 52 months). The 2-year locoregional control (LRC) rate is 77% (median, 61.6 months). The 2-year distant disease free survival (DDFS) rate is 70% (median not reached). There were no differences in radiation-related acute and late toxicities among patients treated pre- versus postoperatively, whether with or without IORT. Conclusions. Compared to surgery alone, neoadjuvant or adjuvant radiation therapy offers patients with RPS an excellent chance for long-term LRC, DDS, and OS. The integration of modern treatment planning for external beam radiation therapy and IORT allows for higher doses to be delivered with acceptable toxicities.
Patients with stage II and III rectal cancer benefit from a multidisciplinary approach to treatment. Studies of postoperative adjuvant therapy consistently demonstrate decreases in locoregional recurrence with the use of radiation therapy. The use of postoperative chemotherapy results in improved disease-free survival and overall survival in certain studies. Preoperative radiation therapy decreases locoregional recurrence and in one study demonstrated an improvement in survival. The addition of chemotherapy to preoperative radiation results in improved locoregional control, but not survival. Preoperative chemoradiation is the standard of care for patients with clinical stage II and III rectal cancer in the United States due to improved local recurrence, acute and late toxicity, and sphincter preservation compared with postoperative chemoradiation. Promising approaches include the incorporation of new chemotherapeutic and biologic agents into chemoradiation and adjuvant chemotherapy regimens; new radiation techniques, such as the use of intraoperative radiation therapy and an accelerated concomitant radiation boost; and gene and protein expression profiling, to better predict response to treatment and prognosis.
Rectal cancer; radiation; chemotherapy; adjuvant; review