A prototype, glassless densitometer traceable to primary optical standards for quantitative radiochromic film dosimetry.

PURPOSE: To evaluate a prototype densitometer traceable to primary optical standards and compare its performance to an EPSON Expression(®) 10000XL flatbed scanner (the Epson) for quantitative radiochromic film (RCF) dosimetry. METHODS: A prototype traceable laser densitometry system (LDS) was developed to mitigate common film scanning artifacts, such as positional scan dependence and high noise in low-dose regions, by performing point-based measurements of RCF suspended in free-space using coherent light. The LDS and the Epson optical absorbance scales were calibrated up to 3 AU, using reference materials calibrated at a primary standards laboratory and a scanner calibration factor (SCF). Calibrated optical density (OD) was determined for 96 Gafchromic(®) EBT3 film segments before and after irradiation to one of 16 dose levels between 0 and 10 Gy, exposed to (60)Co in a polymethyl-methacrylate (PMMA) phantom. The sensitivity was determined at each dose level and at two rotationally orthogonal readout orientations to obtain the sensitometric response of each RCF dosimetry system. LDS rotational scanning dependence was measured at nine angles between 0°and 180°, due to the expected interference between coherent light and polarizing EBT3 material. The response curves were fit to the analytic functions predicted by two physical response models: the two-parameter single-hit model and the four-parameter percolation model. RESULTS: The LDS and the Epson absorbance measurements were linear to primary optical standards to within 0.2% and 0.3% up to 2 and 1 AU, respectively. At higher densities, the LDS had an over-response (2.5% at 3 AU) and the Epson an under-response (3.1% and 9.8% at 2 and 3 AU, respectively). The LDS and the Epson SCF over the applicable range were 0.968% ± 0.2% and 1.561% ± 0.3%, respectively. The positional scan dependence was evaluated on each digitizer and shown to be mitigated on the LDS, as compared to the Epson. Maximum EBT3 rotational dependence was found to have a strong dependence on dose (0.1% and 34% at 30 mGy and 5 Gy, respectively). The preferred EBT3 polymerization axis angle was constant within experimental uncertainties. In its most sensitive orientation, the LDS-measured EBT3 sensitivity was 7.13 × 10(-4) ± 9.2 × 10(-6) AU/mGy, which represented a 4.5 fold increase over the Epson of 1.58 × 10(-4) ± 9.8 × 10(-6) AU/mGy. To first order approximations, EBT3 response was linear up to 500 mGy to within 0.80% and to within 7.5% for the most sensitive LDS and the Epson orientations, respectively. The corresponding single-hit and percolation model relative residual norms were 0.082 and 0.074 for LDS as compared to 0.29 and 0.18 for the Epson, which represented a significant increase in LDS-measured agreement with the simple physical model. Less sensitive LDS and the Epson orientations showed a marked decrease in the physical model agreement, which suggested that suboptimal readout device characteristics may be the origin of the complex sensitometric functional forms currently required for accurate RCF dosimetry. CONCLUSIONS: The prototype densitometer was shown to be superior to a conventional scanner for quantitative RCF dosimetry based on physical models of film response. The Epson was shown to be a reliable tool for routine RCF dosimetry in a clinical setting, yet calibration to primary optical standards did not mitigate the necessity for complex, empirical functional form fitting.

Photon beam audits for radiation therapy clinics: a pilot mailed dosemeter study in Turkey.

A thermoluminescent dosemeter (TLD) mailed dose audit programme was performed at five radiotherapy clinics in Turkey. The intercomparison was organised by the University of Wisconsin Radiation Calibration Laboratory (UWRCL), which was responsible for the technical aspects of the study including reference irradiations, distribution, collection and evaluation. The purpose of these audits was to perform an independent dosimetry check of the radiation beams using TLDs sent by mail. Acrylic holders, each with five TLD chips inside and instructions for their irradiation to specified absorbed dose to water of 2 Gy, were mailed to all participating clinics. TLD irradiations were performed with a 6 MV linear accelerator and (60)Co photon beams. The deviations from the TL readings of UWRCL were calculated. Discrepancies inside the limits of ±5 % between the participant-stated dose, and the TLD-measured dose were considered acceptable. One out of 10 beams checked was outside this limit, with a difference of 5.8 %.

A thermoluminescent dosimetry postal dose inter-comparison of radiation therapy centres in Malaysia.

A thermoluminescent dosimetry (TLD) postal dose inter-comparison was carried out amongst radiotherapy centres in Malaysia. The aim of this TLD inter-comparison was to compare the uniformity involved in the measurement of absorbed dose among the participating centres. A set of 5 TLD chips placed within acrylic trays were mailed to all participating centres for irradiation to an absorbed dose to water of 2 Gy. Measurements were made for 6 MV and 60Co photon beams. Results show an agreement of +/- 5% for all but three radiotherapy centres. The ratios of the TLD readings to that of the reference centre are comparable with other national/regional dose inter-comparisons. The importance of a proper ongoing quality assurance program is essential in maintaining the consistency and uniformity of doses delivered.

Cell cycle regulation of the G0/G1 transition in 5-fluorouracil-sensitive and -resistant human colon cancer cell lines.

PURPOSE: Resistance to 5-fluorouracil (5-FU) has been associated with thymidylate synthase (TS) gene amplification and increased TS protein levels. Increased TS protein expression has also been found to be a significant independent prognostic factor for disease-free survival and overall survival in patients treated with adjuvant 5-FU-based chemotherapy. In these studies and in our prior preclinical studies, TS has been considered a marker of proliferative capacity. The purpose of the current study was to further evaluate the association between TS levels and cell cycle regulation, by investigating cell cycle kinetics in a 5-FU-resistant cell line with constitutive overexpression of TS. The influence of increased TS levels on cell cycle progression may provide insight into methods to overcome 5-FU resistance. MATERIALS: 5-FU-sensitive NCI H630(WT) and 5-FU-resistant NCI H630(R1) (with 15- to 20-fold higher TS protein levels) were utilized in this investigation to determine the influence of constitutive overexpression of TS on cell cycle kinetics. RESULTS: There was no apparent influence of increased TS levels on cell cycle distribution during asynchronous growth, and both cell lines reach plateau growth phase in 120 hours, arresting in G0/G1 as determined by flow cytometry. In the H630(WT) cells, this G0/ G1 arrest was associated with a 14- to 17-fold reduction in TS activity and protein levels (using the TS-106 monoclonal antibody), whereas in the H630(R1) cells, only a two- to fivefold reduction was noted. Flow cytometry analysis utilizing Ki-67 indicated that there was no evidence of a G0 population in the confluent H630(R1), whereas 26% +/- 7% of confluent H630(WT) cells were Ki-67 negative (G0) and the remainder had low Ki-67 signal intensity. Analysis of pRb phosphorylation and p16 and p21 expression suggested that the arrest point for both cell lines was before the point at which Rb phosphorylation takes place, yet the confluent H630(R1) cells had threefold higher p21 than confluent H630(WT) cells. DISCUSSION: These data suggest that the 5-FU-resistant H630(R1) cell lines arrest at a later point in G0/G1 and have a potentially greater capacity for proliferation.

Preclinical toxicity and efficacy study of a 14-day schedule of oral 5-iodo-2-pyrimidinone-2'-deoxyribose as a prodrug for 5-iodo-2'-deoxyuridine radiosensitization in U251 human glioblastoma xenografts.

In anticipation of an initial clinical Phase I trial in patients with high-grade gliomas of p.o. administered 5-iodo2-pyrimidinone-2'-deoxyribose (IPdR) given daily for 14 days as a prodrug for 5-iodo-2'-deoxyuridine (IUdR)-mediated tumor radiosensitization, we determined the systemic toxicities and the percentage IUdR-DNA incorporation in normal athymic mouse tissues and a human glioblastoma xenograft (U251) after this dosing schedule of IPdR. Using a tumor regrowth assay of s.c. U251 xenografts, we also compared radiosensitization with this IPdR-dosing schedule to radiation therapy (XRT) alone (2 Gy/day for 4 days) or to XRT after continuous infusion IUdR for 14 days at the maximum tolerated dose in mice (100 mg/kg/day). Athymic mice with and without U251 s.c. xenografts tolerated 750 or 1500 mg/kg/day of p.o. IPdR (using gastric lavage) for 14 days without weight loss or activity level changes during treatment and for a 28-day posttreatment observation period. The percentage IUdR-DNA incorporation in U251 tumor cells was significantly higher after p.o. IPdR (750 and 1500 mg/kg/day) for 14 days (3.1 +/- 0.2% and 3.7 +/- 0.3%, respectively) than continuous infusion IUdR for 14 days (1.4 +/- 0.1%). Compared to XRT alone, a significant sensitizer enhancement ratio (SER) was found with the combination of p.o. IPdR (1500 mg/kg/d) + XRT (SER = 1.31; P = 0.05) but not for the combination of continuous infusion IUdR + XRT (SER = 1.07; P = 0.57) in the U251 xenografts. The percentage IUdR-DNA incorporation after IPdR at 1500 mg/kg/day for 14 days in normal bone marrow, normal small intestine, and normal liver were 1.2 +/-0.2%, 3.3 +/- 0.3%, and 0.2 +/- 0.1%, respectively. We conclude that a 14-day p.o. schedule of IPdR at up to 1500 mg/kg/day results in no significant systemic toxicity in athymic mice and is associated with significant radiosensitization using this human glioblastoma multiforme xenograft model. Based on these data and our previously published data using shorter IPdR dosing schedules, which also demonstrate an improved therapeutic index for IPdR compared to IUdR, an initial clinical Phase I and pharmacokinetic study of p.o. IPdR daily for 14 days is being designed.

The mismatch repair protein, hMLH1, mediates 5-substituted halogenated thymidine analogue cytotoxicity, DNA incorporation, and radiosensitization in human colon cancer cells.

Deficiency in DNA mismatch repair (MMR) is found in some hereditary (hereditary nonpolyposis colorectal cancer) and sporadic colon cancers as well as other common solid cancers. MMR deficiency has recently been shown to impart cellular resistance to multiple chemical agents, many of which are commonly used in cancer chemotherapy. It is therefore of interest to find an approach that selectively targets cells that have lost the ability to perform MMR. In this study, we examine the response of MMR-proficient (hMLH1+) and MMR-deficient (hMLH1-) colon carcinoma cell lines to the halogenated thymidine (dThd) analogues iododeoxyuridine (IdUrd) and bromodeoxyuridine (BrdUrd) before and after irradiation. These dThd analogues are used clinically as experimental sensitizing agents in radioresistant human cancers, and there is a direct correlation between the levels of dThd analogue DNA incorporation and tumor radiosensitization. In contrast to the well-characterized, marked increase in cytotoxicity (> 1 log cell kill) found with 6-thioguanine exposures in HCT116/3-6 (hMLH1+) cells compared to HCT116 (hMLH1-) cells, we found only modest cytotoxicity (10-20% cell kill) in both cell lines when treated with IdUrd or BrdUrd for 1 population doubling. Upon further analysis, the levels of halogenated dThd analogues in DNA were significantly lower (two to three times lower) in HCT116/3-6 cells than in HCT116 cells, and similar results were found in Mlh1+/+ spontaneously immortalized murine embryonic fibroblasts and fibroblasts from Mlh1 knockout mice. As a result of the higher levels of the dThd analogue in DNA, there was an increase in radiation sensitivity in HCT116 cells but not in HCT116/3-6 cells after pretreatment with IdUrd or BrdUrd when compared to treatment with radiation alone. Additionally, we found no differences in the cellular metabolic pathways for dThd analogue DNA incorporation because the enzyme activities of dThd kinase and thymidylate synthase, as well as the levels of triphosphate pools, were similar in HCT116 and HCT116/3-6 cells. These data suggest that the hMLH1 protein may participate in the recognition and subsequent removal of halogenated dThd analogues from DNA. Consequently, whereas MMR-deficient cells and tumor xenografts have shown intrinsic resistance to a large number of chemotherapeutic agents, the 5-halogenated dThd analogues appear to selectively target such cells for potential enhanced radiation sensitivity.

Preclinical evaluation of 5-iodo-2-pyrimidinone-2'-deoxyribose as a prodrug for 5-iodo-2'-deoxyuridine-mediated radiosensitization in mouse and human tissues.

We reported previously that p.o. administered 5-iodo-2-pyrimidinone-2'-deoxyribose (IPdR) was efficiently converted to 5-iodo-2'-deoxyuridine (IUdR) in athymic mice (T. J. Kinsella et al., Cancer Res., 54: 2695-2700, 1994). Here, we further evaluate IPdR metabolism, systemic toxicity, and percentage DNA incorporation in athymic mouse normal tissues and a human colon cancer xenograft (HT29) using higher p.o. doses of IPdR. These data are compared to results using a continuous infusion of IUdR at the maximum tolerable dose. We also evaluate IPdR metabolism in cytosolic extracts from normal human liver, normal human intestine, and human colorectal cancer specimens. Athymic mice tolerated a daily p.o. bolus of up to 2 g/kg IPdR for 6 days with minimal host toxicity (< or = 10% body weight loss). There was rapid conversion of IPdR to IUdR, with peak plasma levels of IUdR of 40-75 microM at 10 min following a p.o. IPdR bolus of 250-1500 mg/kg. The percentage IUdR-DNA in the HT29 s.c. human tumor xenografts increased 1.5 times (2.3-3.6%) with IPdR doses above 1 g/kg/day for 6 days, whereas the percentage IUdR-DNA incorporation in two proliferating normal tissues (4-4.5% in intestine; 1.6-2.2% in bone marrow) and a quiescent normal tissue (< or = 1% in liver) showed < 1.5-fold increases with the IPdR dose escalation between 1-2 g/kg/day for 6 days. In contrast, using a continuous infusion of IUdR at 100 mg/kg/day, significant systemic toxicity (> 20% body weight loss) was found by day 6 of the infusion. Steady-state plasma IUdR levels were 1.0-1.2 microM during the 6-day infusion, and percentage IUdR-DNA incorporations of 2.3, 8, 6, and 1% were measured in s.c. tumors, normal intestine, normal bone marrow, and normal liver, respectively, following the 6-day infusion. Thus, the p.o. IPdR schedule has an improved therapeutic index, based on percentage IUdR-DNA incorporation in normal and tumor tissues, compared to continuous infusion IUdR at the maximum tolerable dose in athymic mice with this human tumor xenograft. Additionally, a tumor regrowth assay to assess the radiation response of HT29 s.c. xenografts showed a 1.5-fold enhancement (time to regrow to 300% initial tumor volume) with IPdR (1000 mg/kg/day for 6 days) plus fractionated irradiation (XRT; 2 Gy/day for 4 days), compared to XRT (2 Gy/day for 4 days) alone. No enhancement in the radiation response of HT29 s.c. xenografts was found with continuous infusion IUdR (100 mg/kg/day for 6 days) plus XRT (2 Gy/day for 4 days), compared to XRT alone. Using cytosolic extracts from normal human liver specimens, we found a rapid (15-min) conversion of IPdR to IUdR. Coincubation of liver cytosol with IPdR and allopurinol, an inhibitor of xanthine oxidase, had no inhibitory effect on IPdR metabolism, whereas coincubation with IPdR and isovanillin or menadione, analogue substrates for aldehyde oxidase, effectively reduced the amount of IPdR oxidized to IUdR. Significantly less metabolism of IPdR to IUdR was seen in cytosolic extracts from normal human intestine specimens, and no metabolism of IPdR was found in cytosolic extracts from colorectal liver metastases in two patients and from the HT29 human colon cancer xenografts in athymic mice. These additional data indicate that IPdR has the potential for clinical use as a p.o. prodrug for IUdR-mediated radiosensitization of resistant human cancers.

Defective expression of the DNA mismatch repair protein, MLH1, alters G2-M cell cycle checkpoint arrest following ionizing radiation.

A role for the Mut L homologue-1 (MLH1) protein, a necessary component of DNA mismatch repair (MMR), in G2-M cell cycle checkpoint arrest after 6-thioguanine (6-TG) exposure was suggested previously. A potential role for MLH1 in G1 arrest and/or G1-S transition after damage was, however, not discounted. We report that MLH1-deficient human colon carcinoma (HCT116) cells showed decreased survival and a concomitant deficiency in G2-M cell cycle checkpoint arrest after ionizing radiation (IR) compared with genetically matched, MMR-corrected human colon carcinoma (HCT116 3-6) cells. Similar responses were noted between murine MLH1 knockout compared to wild-type primary embryonic fibroblasts. MMR-deficient HCT116 cells or embryonic fibroblasts from MLH1 knockout mice also demonstrated classic DNA damage tolerance responses after 6-TG exposure. Interestingly, an enhanced p53 protein induction response was observed in HCT116 3-6 (MLH1+) compared with HCT116 (MLH1-) cells after IR or 6-TG. Retroviral vector-mediated expression of the E6 protein did not, however, affect the enhanced G2-M cell cycle arrest observed in HCT116 3-6 compared with MLH1-deficient HCT116 cells. A role for MLH1 in G2-M cell cycle checkpoint control, without alteration in G1, after IR was also suggested by similar S-phase progression between irradiated MLH1-deficient and MLH1-proficient human or murine cells. Introduction of a nocodazole-induced G2-M block, which corrected the MLH1-mediated G2-M arrest deficiency in HCT116 cells, clearly demonstrated that HCT116 and HCT116 3-6 cells did not differ in G1 arrest or G1-S cell cycle transition after IR. Thus, our data indicate that MLH1 does not play a major role in G1 cell cycle transition or arrest. We also show that human MLH1 and MSH2 steady-state protein levels did not vary with damage or cell cycle changes caused by IR or 6-TG. MLH1-mediated G2-M cell cycle delay (caused by either MMR proofreading of DNA lesions or by a direct function of the MLH1 protein in cell cycle arrest) may be important for DNA damage detection and repair prior to chromosome segregation to eliminate carcinogenic lesions (possibly brought on by misrepair) in daughter cells.

The interaction of hydroxyurea and ionizing radiation in human cervical carcinoma cells.

PURPOSE: The results from prior in vitro and in vivo studies and recent phase 3 clinical trials suggest a significant potential role for hydroxyurea (HU) as a clinical radiosensitizer for cervix cancer. However, a detailed study of possible cellular mechanisms of radiosensitization in human cervix cancer cells as a consequence of dose and timing of HU and ionizing radiation (IR) has not been performed. This in vitro study analyses the interactions of HU and IR in a human cervical carcinoma cell line, Caski. MATERIALS AND METHODS: Exponentially growing Caski cells were continuously exposed to clinically achievable but minimally cytotoxic concentrations of HU (0.3-3.0 mM) for various time intervals (6, 12, 18, 24, and 30 hours) up to one population doubling time either prior to or immediately following IR (2-8 Gy). The radiation survival data were analyzed using our modification of the linear-quadratic model to test for an interaction (greater than additive). The effects of HU alone, IR alone, and the combination on cell cycle progression and on apoptotic cell death in exponentially growing Caski cells were measured. RESULTS: We report a significant HU-IR interaction (radiosensitization) based on the sequence of HU exposure (post- > pre-IR) and with increasing concentrations of HU (0.3-3.0 mM), but no effect on radiosensitization with the duration of exposure to HU for up to one cell population doubling (6-30 hours). HU concentration has a significant effect on both alpha and beta linear-quadratic values in the post-IR sequences. Exposures of exponentially growing Caski cells to 1 mM and 3 mM HU alone result in a complete block in early S phase throughout the 30-hour exposure, while 0.3 mM HU causes a transient early S-phase block over the initial 12 to 18 hours of exposure. HU alone has no effect on cell cycle progression in G1 or G2/M populations but results in a large apoptotic population (31% following 1 mM HU x 30 hours), which appears to be the principal mechanism of drug cytotoxicity in these cells. IR alone (4 or 6 Gy) results in a significant G2 delay for 6 to 18 hours following IR but no G1 delay and a small apoptotic population at 30 hours post-IR (5.4% vs 2.1% in non-IR controls). The use of HU (0.3 or 1.0 mM) following IR (4 or 6 Gy) results in a significantly larger G2 delay compared with IR alone, but with only an additive effect on the apoptotic population. CONCLUSIONS: These in vitro data demonstrate that radiosensitization of Caski cells is more significant with post-IR exposures to clinically achievable concentrations of HU. This HU-IR interaction is associated with an increased G2 delay, suggesting a reduction in IR damage repair. However, this interaction appears to be independent of the cytotoxicity (principally by apoptosis) from HU alone.

Leucovorin modulation of 5-iododeoxyuridine radiosensitization: a phase I study.

Evidence for clinically significant radiosensitization by the halogenated pyrimidine 5-iododeoxyuridine (IdUrd) continues to accumulate. In vitro radiosensitization has been demonstrated in human colon tumor cell lines following exposure to 1-10 micrometer. Coadministration of leucovorin (LV) increases radiosensitization, which correlates directly with increased IdUrd DNA incorporation. Clinical data regarding proliferation rates and thymidine kinase levels in tumors versus normal tissues suggest selective incorporation of IdUrd into gastrointestinal tumors may occur. The objectives of this Phase I study were: (a) to assess the feasibility of LV modulation of IdUrd radiosensitization by determining the maximum tolerated dose (MTD) of IdUrd plus LV; and (b) to perform correlative laboratory studies to investigate the potential of IdUrd plus LV to increase radiosensitization in vivo. Seventeen patients with unresectable or recurrent gastrointestinal adenocarcinomas received a 14-day course of continuous i.v. infusion of IdUrd prior to initiation of radiotherapy. Two additional 14-day infusions of IdUrd with LV were given during the course of radiotherapy (60 Gy in 6 weeks). The initial dose of IdUrd was 250 mg/m2/day and was escalated in subsequent patients to 400 and 600 mg/m2/day. The LV dose remained fixed at 250 mg/m2/day. Leukopenia was the dose-limiting toxicity, and 400 mg/m2/day was the MTD for this trial. At the MTD, the mean +/- SD steady-state plasma concentration of IdUrd during the infusion, measured by high-performance liquid chromatography, was 0.66 +/- 0.23 micrometer. There was no significant influence of LV on IdUrd DNA incorporation in peripheral blood granulocytes as measured by high-performance liquid chromatography. Based on toxicity data and correlative laboratory studies, a meaningful increase in radiosensitization would not be achieved with the IdUrd infusion schedule and dose of LV investigated compared with IdUrd alone.

In vivo modulation of iododeoxyuridine metabolism and incorporation into cellular DNA by 5'-amino-5'-deoxythymidine in normal mouse tissues and two human colon cancer xenografts.

This in vivo study examines the ability of 5'-amino-5'-deoxythymidine (5'-AdThd) to modulate 5-iododeoxyuridine (IdUrd) cellular metabolism in two human colon cancer xenografts (HT 29 and HCT-116), two actively proliferating normal mouse tissues (bone marrow and intestine), and a quiescent normal mouse tissue (liver). 5'-AdThd is a thymidine analogue that at low concentrations (<30 micrometer) can increase thymidine kinase activity, which is the rate-limiting enzyme for activation of IdUrd. We reported recently that the in vitro incubation of HT 29 and HCT-116 cells in 5'-AdThd + IdUrd resulted in an enhancement of 5-iodo-2'-dUTP pools, IdUrd DNA incorporation, and subsequent radiosensitization compared with incubation with IdUrd alone (Clin. Cancer Res., 1: 407-416, 1995). These in vitro effects were more significant in the radioresistant cell line HT 29. Using a 6-day continuous infusion of IdUrd (50 or 100 mg/kg/day) and/or 5'-AdThd (200 mg/kg/day), no increase in systemic toxicity (percentage of body weight loss) was observed in athymic nude mice with 5'-AdThd alone or when combined with IdUrd. There was significant dose-dependent, systemic toxicity with IdUrd, which was reversible within 3 days of completing the lower-dose IdUrd infusion. However, a comparison of plasma levels during the 6-day continuous infusion of IdUrd +/- 5'-AdThd showed a significant interaction of IdUrd and 5'-AdThd, resulting in higher plasma levels by day 6 of both compounds and the principal metabolites, iodouracil and deoxyuridine, which is consistent with nonlinear saturating effects on dihydrouracil dehydrogenase. Coadministration of IdUrd and 5'-AdThd resulted in an increase in the percentage of IdUrd DNA incorporation in the two proliferating normal tissues, which was significant only with the lower IdUrd dose. No effect on IdUrd DNA incorporation was found in normal liver at either IdUrd dose +/- 5'-AdThd. Similar to our in vitro data, the continuous infusion of IdUrd and 5'-AdThd showed a significant effect by increasing the percentage of IdUrd DNA incorporation in HT-29 xenografts at both IdUrd doses, whereas coadministration of 5'-AdThd had no such effect in HCT-116 xenografts.

The interaction of hydroxyurea and iododeoxyuridine on the radiosensitivity of human bladder cancer cells.

Biochemical modulation of iododeoxyuridine (IdUrd) incorporation into the DNA of tumor cells is a potential clinical strategy to enhance radiosensitivity and to simultaneously differentiate the sensitivity of rapidly proliferating tumor cells and more slowly proliferating adjacent normal tissues to radiation. The interactions of hydroxyurea (HU) and IdUrd were studied in a human bladder cancer cell line, 647V. Exposure of exponentially growing 647V cells to HU concentrations of 10-100 microM for one cell population doubling (24 h) resulted in no cytotoxicity as assessed by clonogenic survival. Flow cytometric analysis showed a significant increase in an early S-phase population after a 12-h exposure but a return to a normal cell cycle distribution after a 24-h exposure to 100 microM HU. Incorporation of IdUrd into DNA was increased 2-fold by coincubation with HU (100 microM) and a clinically achievable concentration of IdUrd (2 microM) for 24 h. To elucidate the mechanism of modulation, IdUTP pools were compared in 647V cells treated with 2 microM IdUrd with or without 100 microM HU. A 2-fold increase in IdUTP pools was evident within 2 h when this drug combination was used. With the use of multivariate statistical analysis, the radiosensitivity of 647V cells was compared after a 24-h exposure to various concentrations of IdUrd (0 and 2 microM) and HU (0, 10, and 100 microM). A 24-h exposure to 100 microM HU alone or to 2 microM IdUrd alone before irradiation resulted in significant (P < 0.02) radiosensitization with sensitizer enhancement ratios of 1.15 and 1.27, respectively. A 24-h exposure to 100 microM HU + 2 microM IdUrd resulted in even more significant (P = 0.001) radiosensitization, which was found to be a greater than additive response (sensitizer enhancement ratio, 1.76 observed compared with 1.37 expected). No radiosensitization was found with a 12-h exposure to 100 microM HU alone. The mechanism of biochemical modulation of IdUrd by a noncytotoxic dose of HU is proposed as increasing the IdUTP pools by stimulating enzymes in the thymidine salvage pathway and subsequently enhancing IdUrd incorporation and radiosensitization.

[Mass-screening for ovarian cancer by transvaginal ultrasonography--study on ultrasonographic findings].

Mass-screening for ovarian cancer by means of transvaginal ultrasonography has been performed in Aomori Prefecture since 1989. To select appropriate candidates to receive the second screening, the findings in 614 ultrasonic pictures of pelvic tumors over 30mm recorded on a VTR were studied. The results were as follows: 1) At the first screening, the rate of solid tumors was similar in each age group, but that of mixed tumors was higher in the thirties. 2) When ultrasonic findings at the first screening with that of the second screening were compared, (1) Twenty to thirty per cent of all tumors detected in the first screening had disappeared or had become under 30mm in size at the second screening. (2) About 20% of solid tumors detected in the first screening were not ovarian tumors (uterine myomas or others) in the second screening, regardless of the tumor size. (3) The rate which decided the course of therapy or follow up at the time of the second screening was not related to the ultrasonic findings for tumors smaller than 50mm. At the present time, classification of transvaginal ultrasonic findings is insufficient, and so it is difficult to use it to select appropriate candidates to receive the second screening. But the ultrasonic findings are very important in deciding on the course of therapy and follow up.

The role of cell cycle redistribution in radiosensitization: implications regarding the mechanism of fluorodeoxyuridine radiosensitization.

PURPOSE: Radiosensitization has previously been demonstrated in a human colon cancer cell line (HT-29) following a 2 h exposure to low, clinically relevant concentrations (0.05-0.5 microM) of fluorodeoxyuridine (FdUrd) (15). The sensitizer enhancement ratio value (measured at 10% survival) plateaued at approximately 1.7 between 16 and 32 h following removal of drug. Parallel studies investigating the effect of FdUrd on the distribution of cells throughout the cell cycle found that the percentage of cells in early S-phase increased to approximately 70% during the same period that maximal radiosensitization was noted. As a follow-up to these findings, experiments have been designed to investigate the contribution of this early S-phase delay to radiosensitization. METHODS AND MATERIALS: Synchronized populations of HT-29 cells have been obtained with three separate techniques. Two involve the induction of a reversible metaphase arrest (with high pressure N2O or colcemid) followed by a shakeoff of mitotic cells. The third uses a plant amino acid, mimosine, to induce a reversible block at the G1/S boundary. Flow cytometry was used to analyze the degree of synchrony based on bromodeoxyuridine (BrdUrd) uptake and propidium iodide (PI) staining. Radiation survival curves were obtained on these synchronized populations to investigate changes in radiosensitivity through the cell cycle. Additionally, levels of thymidylate synthase (TS), the primary target of FdUrd cytotoxicity, were measured in each phase of the cell cycle using the TS 106 monoclonal antibody against human TS. RESULTS: Synchronization with mitotic shakeoff produced relatively pure populations of cells in G1; however, the degree of synchrony in early S-phase was limited both by cells remaining in G1 and by cells progressing into late S-phase. These techniques failed to reveal increased radiosensitivity in early S-phase at 10% survival. An 18 h exposure to mimosine resulted in populations that more closely resembled the early S-phase enrichment following FdUrd exposure and revealed increased radiosensitivity during early S-phase. TS levels were noted to be only 1.3 times higher in S phase than in G0/G1. CONCLUSION: Radiation survival data from cells synchronized with mitotic shakeoff techniques suggest that early S-phase delay is unlikely to be the primary mechanism of FdUrd radiosensitization. In contrast, the increased sensitivity seen in early S-phase with mimosine synchronized cells is similar to that seen with FdUrd. Although confounding biochemical pertubations cannot be ruled out, these data continue to suggest an association between early S-phase enrichment and radiosensitization. The significance of TS inhibition as a mechanism of FdUrd radiosensitization remains unclear.

An in vivo comparison of oral 5-iodo-2'-deoxyuridine and 5-iodo-2-pyrimidinone-2'-deoxyribose toxicity, pharmacokinetics, and DNA incorporation in athymic mouse tissues and the human colon cancer xenograft, HCT-116.

5-iodo-2-pyrimidinone-2'-deoxyribose (IPdR) was recently reported to be converted to 5-iodo-2'-deoxyuridine (IUdR) by an aldehyde oxidase, most concentrated in liver tissue. We questioned whether IPdR could be used as a p.o. hepatotropic prodrug to increase the percentage of IUdR-DNA incorporation into liver tumors compared to normal liver with acceptable systemic toxicity. Athymic nude mice with human colon cancer (HCT-116) xenograft tumors as liver metastases and s.c. flank tumors received daily p.o. boluses (via gastric tubes) of IUdR or IPdR for 6 days. The maximum tolerated dose of IUdR was 250 mg/kg/day and was associated with a > 10% weight loss and a high percentage of IUdR-DNA incorporation (> 5%) into normal bone marrow and intestine. In contrast, animals tolerated escalating doses of IPdR to 1 gm/kg/day without weight loss and with less (1.5-4%) IUdR-DNA incorporation in normal tissues. Pharmacokinetic analysis of p.o. IPdR showed peak plasma levels of IPdR and IUdR within 15-45 min, suggesting efficient conversion of IPdR to IUdR. Aldehyde oxidase activity was found in normal liver tissue but not in other normal or tumor tissues. Additionally, we found a 2-3 times greater percentage of IUdR-DNA incorporation in tumor with IPdR than IUdR at the highest doses used. However, no differential effect in the percentage of IUdR-DNA incorporation was noted between liver metastases and s.c. tumors with either IPdR or IUdR. We conclude that p.o. IPdR offers a greater therapeutic index for tumor incorporation (and presumably radiosensitization) than a similar schedule of IUdR.

Iododeoxyuridine chemosensitization of cis-diamminedichloroplatinum(II) in human bladder cancer cells.

We investigated the effect of iododeoxyuridine (IdUrd) exposure on cis-diamminedichloroplatinum (CDDP) cytotoxicity in the human bladder cancer cell line 647V. Following a 48-h incubation with 2-20 microM IdUrd, a 1-h exposure to 0-120 microM CDDP produced a dose-dependent increase in CDDP cytotoxicity as measured by clonogenic survival. IdUrd exposure of 2, 5, 10, and 20 microM prior to CDDP resulted in dose-modifying factors at 10% survival of 1.2, 1.6, 2.0, and 3.5, respectively. The increase in CDDP cytotoxicity appears to be associated with the level of DNA thymidine replacement in DNA by IdUrd over the range of 13-36%. Atomic absorption spectrophotometric analysis of DNA extracted from 647V cells showed that IdUrd substitution did not affect the total amount of platinum bound to the DNA or the persistence of the bound platinum over a 24-h period post-CDDP exposure versus control cells. IdUrd, unlike thymidine, was found to form two monofunctional adducts with CDDP both in vitro and in vivo. IdUrd was also found to form a mixed bifunctional adduct with deoxyguanosine (dGua) and CDDP in vitro. 1H NMR analysis of purified IdUrd-Pt and IdUrd-Pt-dGua adducts confirmed the identity of these adducts. High pressure liquid chromatography analysis of [3H]IdUrd-labeled 647V DNA digests exposed to CDDP showed the presence of two monofunctional adducts. Unlike the free solution production of adducts in vitro, the predominant adduct formed was not IdUrd-Pt. Results using 125IdUrd-labeled 647V DNA suggests that this adduct is 5-Pt-deoxyuridine. We were not able, however, to detect the presence of the bifunctional adducts IdUrd-Pt-dGua or dUrd-Pt-dGua. This was most likely due to the extremely low proportion of mixed bifunctional adducts produced in vivo. Nonetheless, these results suggest that IdUrd DNA incorporation may enhance CDDP cytotoxicity through the increase of available sites for Pt adduct formation. A Phase I clinical trial of this approach is planned.

Effects of bleomycin with iododeoxyuridine, 5'-amino-5'-deoxythymidine, and radiation in a human bladder cancer cell line.

In vitro bleomycin cytotoxicity in 647V, a human bladder cancer cell line, is enhanced when the cells are preincubated for one cell cycle (24 h) with clinically relevant concentrations (2 microM) of iododeoxyuridine (IdUrd). Bleomycin cytotoxicity after a 1-h exposure is further enhanced by a 24-h preincubation with 2 microM IdUrd plus 30 microM 5'-amino-5'-deoxythymidine (5'-AdThd). Chemosensitization of clinically achievable plasma levels of bleomycin (1-10 mU/ml for 1 h) by IdUrd (+/- 5'-AdThd) is associated with the level of DNA incorporation of IdUrd in 647V cells and with enhanced bleomycin-induced damage in IdUrd-substituted DNA compared to control DNA, as measured by rapid alkaline elution. We also studied the in vitro effects of bleomycin (+/- IdUrd) treatment on the subsequent radiation response of 647V cells. We found that a 1-h preirradiation exposure of 647V cells to low concentrations of bleomycin (1 mU/ml) can radiosensitize the cells with an even greater interaction exhibited by pretreatment with 2 microM IdUrd (24 h) plus bleomycin (1 h) vs control. Since the principal normal tissue toxicities of IdUrd (involving bone marrow and gut) and bleomycin (involving lung) do not overlap, these in vitro data suggest that chemosensitization of bleomycin by IdUrd may provide a new therapeutic regimen for chemotherapy and a significant enhancement for radiotherapy in human bladder cancer and possibly other epithelial cancers.

Global comparison of radiation and chemotherapy dose-response curves with a test for interaction.

In the analysis of the effects of radiation or drugs on clonogenic survival data of mammalian cells, it is often advantageous to compare entire dose-response curves generated under different experimental conditions rather than to conduct single-dose comparisons. We propose a two-stage method for the global comparison of such curves. The first stage consists of individual fits of a flexible model to the dose-response sequences. The second stage treats the fitted coefficients as data and analyzes them jointly using a multivariate analysis of variance. For dose-response models of the type commonly used in the analysis of clonogenic survival data, this method allows the definition of a statistical test for interaction among treatments. That is, a test that the combination of agents produces a dose-response curve which is not what would be expected if the agents were acting multiplicatively (additively in the log cell survival scale). Analyses of cell survival curves by this method for experiments on iododeoxyuridine-mediated radiosensitization and on chemosensitization of bleomycin cytotoxicity in a human bladder cancer cell line (647V) are presented.

Low pH does not affect the dose response for 5'-amino-5'-deoxythymidine modulation of IdUrd DNA incorporation and radiosensitization in a human bladder cancer cell line.

We report that coincubation of 647V cells for one cell cycle with low concentrations (30 microM) of 5'-amino-5'-deoxythymidine increased IdUrd DNA incorporation and radiosensitivity at low extracellular pH (pHe 6.8) in a fashion similar to treatment at normal pHe. IdUrd DNA incorporation is inhibited by high (300 microM) 5'-AdThd concentrations at both normal and low pHe (7.4 and 6.8), resulting in no significant radiosensitization. These results at low pHe were not anticipated based on previously published studies of 5'-AdThd modulation of thymidine kinase (TK) activity and nucleoside cellular uptake. Our results suggest that regulation of intracellular pH (pHi) during the course of one cell cycle negates the 5'-AdThd dose-dependent modulation of TK activity demonstrated previously. Flow cytometric measurement of pHi in 647V cells showed that normal pHi (pH 7.4) was maintained in 647V cells over a 12- to 24-h exposure to low pHe (pH 6.8). Thus the concomitant use of IdUrd and high concentrations of 5'-AdThd (> 30 microM) is unlikely to result in selective in vivo radiosensitization of human tumors under conditions which are intermittently or chronically acidic. However, low concentrations of 5'-AdThd may prove to be an effective in vivo modulator of IdUrd radiosensitization of human tumors under both normal and acidic conditions.

Modulation of IdUrd-DNA incorporation and radiosensitization in human bladder carcinoma cells.

5' Amino-5'-deoxythymidine (5'-AdThd) has been demonstrated previously to antagonize dTTP-mediated feedback inhibition of purified thymidine kinase from 647V, a human bladder cancer cell line. Low concentrations of 5'-AdThd (3-30 microM) have also been shown to stimulate cellular uptake of iododeoxyuridine (IdUrd) in 647V cells at clinically relevant IdUrd concentrations (2 microM). We report that the combination of 30 microM 5'-AdThd plus 2 microM IdUrd results in a significant increase of IdUrd replacement of thymidine (dThd) (18%) in the DNA of 647V cells over that obtained by exposure to 2 microM IdUrd alone (7.9%). However, increasing the 5'-AdThd concentration to 300 microM inhibited the incorporation of IdUrd into DNA (3%). IdUrd-induced radiosensitization of 647V cells, as measured by clonogenic survival, was enhanced by coincubation with 30 microM 5'-AdThd, while 300 microM 5'-AdThd reduced the IdUrd radiosensitization. Additionally, radiation-induced single strand break generation when IdUrd was incorporated into 647V DNA, as measured by rapid alkaline elution, was also enhanced by coincubation with 30 microM 5'-AdThd, while 300 microM 5'-AdThd resulted in a decrease in the number of single strand breaks produced. In T24, another bladder cancer cell line, and SV-HUC-TT1, a tumorigenic cell line derived from SV-HUC, 3-10 microM 5'-AdThd was also able to enhance IdUrd replacement of dThd in DNA. However, no stimulation of dThd replacement by 5'-AdThd occurred in SV-HUC, a prototypic "normal" bladder urothelial cell line. Since 5'-AdThd is not a substrate for mammalian thymidine kinase and has little or no cytotoxicity in vitro and in vivo, it may be a selective modulator of IdUrd radiosensitization of human bladder carcinoma and should be tested in vivo.