Quality of coverage: conformity measures for stereotactic radiosurgery.

In radiosurgery, conformity indices are often used to compare competing plans, evaluate treatment techniques, and assess clinical complications. Several different indices have been reported to measure the conformity of the prescription isodose to the target volume. The PITV recommended in the Radiation Therapy Oncology Group (RTOG) radiosurgery guidelines, defined as the ratio of the prescription isodose volume (PI) over the target volume (TV), is probably the most frequently quoted. However, these currently used conformity indices depend on target size and shape complexity. The objectives of this study are to systematically investigate the influence of target size and shape complexity on existing conformity indices, and to propose a different conformity index-the conformity distance index (CDI). The CDI is defined as the average distance between the target and the prescription isodose line. This study examines five case groups with volumes of 0.3, 1.0, 3.0, 10.0, and 30.0 cm(3). Each case group includes four simulated shapes: a sphere, a moderate ellipsoid, an extreme ellipsoid, and a concave "C" shape. Prescription dose coverages are generated for three simplified clinical scenarios, i.e., the PI completely covers the TV with 1 and 2 mm margins, and the PI over-covers one half of the TV with a 1 mm margin and under-covers the other half with a 1 mm margin. Existing conformity indices and the CDI are calculated for these five case groups as well as seven clinical cases. When these values are compared, the RTOG PITV conformity index and other similar conformity measures have much higher values than the CDI for smaller and more complex shapes. With the same quality of prescription dose coverage, the CDI yields a consistent conformity measure. For the seven clinical cases, we also find that the same PITV values can be associated with very different conformity qualities while the CDI predicts the conformity quality accurately. In summary, the proposed CDI provides more consistent and accurate conformity measurements for all target sizes and shapes studied, and therefore will be a more useful conformity index for irregularly shaped targets.

Mobile linear accelerators for intraoperative radiation therapy.

Intraoperative radiation therapy (IORT) is becoming an increasingly common procedure for treating gross tumors or tumor beds after resection. Traditionally, IORT delivery required either heavily shielded ORs or transporting anesthetized patients to the department of radiation oncology. The availability of a self-shielded mobile electron linear accelerator has made this treatment modality accessible to institutions that otherwise would not consider performing IORT. This article describes IORT equipment and supplies and addresses perioperative nursing issues, as well as the roles of other team members involved in the delivery of IORT.

In vivo dosimetry using a single diode for megavoltage photon beam radiotherapy: implementation and response characterization.

The AAPM Task Group 40 reported that in vivo dosimetry can be used to identify major deviations in treatment delivery in radiation therapy. In this paper, we investigate the feasibility of using one single diode to perform in vivo dosimetry in the entire radiotherapeutic energy range regardless of its intrinsic buildup material. The only requirement on diode selection would be to choose a diode with the adequate build up to measure the highest beam energy. We have tested the new diodes from Sun Nuclear Corporation (called QED and ISORAD-p--both p-type) for low-, intermediate-, and high-energy range. We have clinically used both diode types to monitor entrance doses. In general, we found that the dose readings from the ISORAD (p-type) are closer of the dose expected than QED diodes in the clinical setting. In this paper we report on the response of these newly available ISORAD (p-type) diode detectors with respect to certain radiation field parameters such as source-to-surface distance, field size, wedge beam modifiers, as well as other parameters that affect detector characteristics (temperature and detector-beam orientation). We have characterized the response of the high-energy ISORAD (p-type) diode in the low- (1-4 MV), intermediate- (6-12 MV), and high-energy (15-25 MV) range. Our results showed that the total variation of the response of high-energy ISORAD (p-type) diodes to all the above parameters are within +/-5% in most encountered clinical patient treatment setups in the megavoltage photon beam radiotherapy. The usage of the high-energy buildup diode has the additional benefit of amplifying the response of the diode reading in case the wrong energy is used for patient treatment. In the light of these findings, we have since then switched to using only one single diode type, namely the "red" diode; manufacturer designation of the ISORAD (p-type) high-energy (15-25 MV) range diode, for all energies in our institution and satellites.

Loss of DNA mismatch repair imparts defective cdc2 signaling and G(2) arrest responses without altering survival after ionizing radiation.

Our previous data demonstrated that cells deficient in MutL homologue-1 (MLH1) expression had a reduced and shorter G(2) arrest after high-dose-rate ionizing radiation (IR), suggesting that the mismatch re pair (MMR) system mediates this cell cycle checkpoint. We confirmed this observation using two additional isogenetically matched human MLH1 (hMLH1)-deficient and -proficient human tumor cell systems: human ovarian cancer cells, A2780/CP70, with or without ectopically expressed hMLH1, and human colorectal carcinoma cells, RKO, with or without azacytidine treatment to reexpress hMLH1. We also examined matched MutS homologue-2 (hMSH2)-deficient and -proficient human endometrial carcinoma HEC59 cell lines to determine whether hMSH2, and MMR in general, is involved in IR-related G(2) arrest responses. As in MLH1-deficient cells, cells lacking hMSH2 demonstrated a similarly altered G(2) arrest in response to IR (6 Gy). These differences in IR-induced G(2) arrest between MMR-proficient and -deficient cells were found regardless of whether synchronized cells were irradiated in G(0)/G(1) or S phase, indicating that MMR indeed dramatically affects the G(2)-M checkpoint arrest. However, unlike the MMR-dependent damage tolerance response to 6-thioguanine exposures, no significant difference in the clonogenic survival of MMR-deficient cells compared with MMR-proficient cells was noted after high-dose-rate IR. In an attempt to define the signal transduction mechanisms responsible for MMR-mediated G(2) arrest, we examined the levels of tyrosine 15 phosphorylation of cdc2 (phospho-Tyr15-cdc2), a key regulator of the G(2)-M transition. Increased phospho-Tyr15-cdc2 levels were observed in both MMR-proficient and -deficient cell lines after IR. However, the levels of the phospho-Tyr15-cdc2 rapidly decreased in MMR (hMLH1 or hMSH2)-deficient cell lines at times coincident with progress from the IR-induced G(2) arrest through M phase. Thus, differences in the levels of phospho-Tyr15-cdc2 after high-dose-rate IR correspond temporally with the observed differences in the IR-induced G(2) arrest, suggesting that MMR proteins may exert their effect on IR-induced G(2) arrest by signaling the cdc2 pathway. Although MMR status does not significantly affect the survival of cells after high-dose-rate IR, it seems to regulate the G(2)-M checkpoint and might affect overall mutation rates.

Photodynamic therapy in oncology.

Photodynamic therapy (PDT) is a cancer treatment modality that is based on the administration of a photosensitiser, which is retained in tumour tissues more than in normal tissues, followed by illumination of the tumour with visible light in a wavelength range matching the absorption spectrum of the photosensitiser. The photosensitiser absorbs light energy and induces the production of reactive oxygen species in the tumour environment, generating a cascade of events that kills the tumour cells. The first generation photosensitiser, Photofrin (porfirmer sodium), has been approved for oesophageal and lung cancer in the US and has been under investigation for other malignant and non-malignant diseases. Sub-optimal light penetration at the treatment absorption peak of Photofrin and prolonged skin photosensitivity in patients are limiting factors for this preparation. Several new photosensitisers have improved properties, especially absorption of longer wavelength light which penetrates deeper into tissue and faster clearance from normal tissue. This paper reviews the current use of first- and second-generation photosensitisers in oncology. The use of PDT in oncology has been restricted to certain cancer indications and has not yet become an integral part of cancer treatment in general. The main advantage of PDT is that the treatment can be repeated multiple times safely, without producing immunosuppressive and myelosuppressive effects and can be administered even after surgery, chemotherapy or radiotherapy. The current work on new photosensitisers and light delivery equipment will address some of the present shortcomings of PDT. Much has been learned in recent years about the mechanisms of cellular and tissue responses to PDT and protocols designed to capitalise on this knowledge showed lead to additional improvements.

Methoxyamine potentiates DNA single strand breaks and double strand breaks induced by temozolomide in colon cancer cells.

We have previously shown that human cancer cells deficient in DNA mismatch repair (MMR) are resistant to the chemotherapeutic methylating agent temozolomide (TMZ) and can be sensitized by the base excision repair (BER) blocking agent methoxyamine (MX) [21]. To further characterize BER-mediated repair responses to methylating agent-induced DNA damage, we have now evaluated the effect of MX on TMZ-induced DNA single strand breaks (SSB) by alkaline elution and DNA double strand breaks (DSB) by pulsed field gel electrophoresis in SW480 (O6-alkylguanine-DNA-alkyltransferase [AGT]+, MMR wild type) and HCT116 (AGT+, MMR deficient) colon cancer cells. SSB were evident in both cell lines after a 2-h exposure to equitoxic doses of temozolomide. MX significantly increased the number of TMZ-induced DNA-SSB in both cell lines. In contrast to SSB, TMZ-induced DNA-DSB were dependent on MMR status and were time-dependent. Levels of 50 kb double stranded DNA fragments in MMR proficient cells were increased after TMZ alone or in combination with O6-benzylguanine or MX, whereas, in MMR deficient HCT116 cells, only TMZ plus MX produced significant levels of DNA-DSB. Levels of AP endonuclease, XRCC1 and polymerase beta were present in both cell lines and were not significantly altered after MX and TMZ. However, cleavage of a 30-mer double strand substrate by SW480 and HCT116 crude cell extracts was inhibited by MX plus TMZ. Thus, MX potentiation of TMZ cytotoxicity may be explained by the persistence of apurinic/apyrimidinic (AP) sites not further processed due to the presence of MX. Furthermore, in MMR-deficient, TMZ-resistant HCT116 colon cancer cells, MX potentiates TMZ cytotoxicity through formation of large DS-DNA fragmentation and subsequent apoptotic signalling.

Targeting DNA mismatch repair for radiosensitization.

Postreplicational mismatch repair (MMR) proteins are capable of recognizing and processing not only single base-pair mismatches and insertion-deletion loops (IDLs) that occur during DNA replication, but also adducts in DNA resulting from treatment with cancer chemotherapy agents. These include widely varying types of DNA adducts resulting from methylating agents such as MNNG, MNU, temozolomide, and procarbazine; CpG crosslinks resulting from cisplatin and carboplatin; and S(6)-thioguanine and S(6)-methylthioguanine residues in DNA. Although MMR proteins can recognize both replicational errors and chemotherapy-induced adducts in DNA, the end results of this recognition are very different. Base-base mismatches and IDLs can be repaired by MMR, restoring genomic integrity, whereas MMR-mediated recognition and processing of chemotherapy-induced adducts in DNA results in apoptosis. After the loss of MMR, the inability of cells to recognize and correct single base-pair mismatches and insertion-deletion loops can lead to secondary mutations in proto-oncogenes and tumor-suppressor genes, thereby contributing to the development of cancer. In addition, the inability of MMR-deficient cells to recognize chemotherapy-induced adducts in DNA can result in a damage-tolerant phenotype that translates to clinically significant resistance by allowing for selection of MMR-deficient cancer cells. We have shown recently that these MMR-deficient, drug-resistant cells can be targeted for radiosensitization by the halogenated thymidine analogs iododeoxyuridine (IdUrd) and bromodeoxyuridine (BrdUrd). These thymidine (dThd) analogs become incorporated into DNA and form reactive uracil radicals after ionizing radiation (IR), increasing strand breaks. IdUrd and BrdUrd appear to be removed from DNA in MMR-proficient cells with limited toxicity or disruption of the cell cycle, while accumulating at much higher levels in MMR-deficient cells. As a result, it is possible to effectively increase the radiosensitization of MMR-deficient cells at levels of halogenated dThd analog that demonstrate limited toxicity to MMR-proficient cells. This indicates that a combined approach of IdUrd or BrdUrd with IR may be effective in killing MMR-deficient tumors in patients, which are resistant to many cancer chemotherapy agents commonly used in the clinic.

When X-ray-inducible proteins meet DNA double strand break repair.

Cellular responses to ionizing radiation (IR) include (a) activation of signal transduction enzymes; (b) stimulation of DNA repair, most notably DNA double strand break (DSB) repair by homologous or nonhomologous recombinatorial pathways; (c) activation of transcription factors and subsequent IR-inducible transcript and protein changes; (d) cell cycle checkpoint delays in G(1), S, and G(2) required for repair or for programmed cell death of severely damaged cells; (e) activation of zymogens needed for programmed cell death (although IR is a poor inducer of such responses in epithelial cells); and (f) stimulation of IR-inducible proteins that may mediate bystander effects influencing signal transduction, DNA repair, angiogenesis, the immune response, late responses to IR, and possibly adaptive survival responses. The overall response to IR depends on the cell's inherent genetic background, as well as its ability to biochemically and genetically respond to IR-induced damage. To improve the anti-tumor efficacy of IR, our knowledge of these pleiotropic responses must improve. The most important process for the survival of a tumor cell following IR is the repair of DNA double strand breaks (DSBs). Using yeast two-hybrid analyses along with other molecular and cellular biology techniques, we cloned transcripts/proteins that are involved in, or presumably affect, nonhomologous DNA double strand break end-joining (NHEJ) repair mediated by the DNA-PK complex. Using Ku70 as bait, we isolated a number of Ku-binding proteins (KUBs). We identified the first X-ray-inducible transcript/protein (xip8, Clusterin (CLU)) that associates with DNA-PK. A nuclear form of CLU (nCLU) prevented DNA-PK-mediated end joining, and stimulated cell death in response to IR or when overexpressed in the absence of IR. Structure-function analyses using molecular and cellular (including green fluorescence-tagged protein trafficking) biology techniques showed that nCLU appears to be an inactive protein residing in the cytoplasm of epithelial cells. Following IR injury, nCLU levels increase and an as yet undefined posttranslational modification appears to alter the protein, exposing nuclear localization sequences (NLSs) and coiled-coil domains. The modified protein translocates to the nucleus and triggers cell death, presumably through its interaction specifically with Ku70. Understanding nCLU responses, as well as the functions of the KUBs, will be important for understanding DSB repair. Knowledge of DSB repair may be used to improve the antitumor efficacy of IR, as well as other chemotherapeutic agents.

A new approach to intraoperative radiation therapy.

Intraoperative radiation therapy (IORT) is an adjuvant treatment in which a large single dose of radiation is delivered during a surgical procedure to resected tumor beds or to an unresectable tumor. This article discusses the implementation of an IORT program and highlights the successful collaboration needed between the OR and radiation oncology departments. A better understanding of IORT in the OR setting will contribute to smooth program implementation.

Role of the hMLH1 DNA mismatch repair protein in fluoropyrimidine-mediated cell death and cell cycle responses.

DNA mismatch repair (MMR) is an efficient system for the detection and repair of mismatched and unpaired bases in DNA. Deficiencies in MMR are commonly found in both hereditary and sporadic colorectal cancers, as well as in cancers of other tissues. Because fluorinated thymidine analogues (which through their actions might generate lesions recognizable by MMR) are widely used in the treatment of colorectal cancer, we investigated the role of MMR in cellular responses to 5-fluorouracil and 5-fluoro-2'-deoxyuridine (FdUrd). Human MLH1(-) and MMR-deficient HCT116 colon cancer cells were 18-fold more resistant to 7.5 microM 5-fluorouracil (continuous treatment) and 17-fold more resistant to 7.5 microM FdUrd in clonogenic survival assays compared with genetically matched, MLH1(+) and MMR-proficient HCT116 3-6 cells. Likewise, murine MLH1(-) and MMR-deficient CT-5 cells were 3-fold more resistant to a 2-h pulse of 10 microM FdUrd than their MLH1(+) and MMR-proficient ME-10 counterparts. Decreased cytotoxicity in MMR-deficient cells after treatment with various methylating agents and other base analogues has been well reported and is believed to reflect a tolerance to DNA damage. Synchronized HCT116 3-6 cells treated with a low dose of FdUrd had a 2-fold greater G(2) cell cycle arrest compared with MMR-deficient HCT116 cells, and asynchronous ME-10 cells demonstrated a 4-fold greater G(2) arrest after FdUrd treatment compared with CT-5 cells. Enhanced G(2) arrest in MMR-proficient cells in response to other agents has been reported and is believed to allow time for DNA repair. G(2) cell cycle arrest as determined by propidium iodide staining was not a result of mitotic arrest, but rather a true G(2) arrest, as indicated by elevated cyclin B1 levels and a lack of staining with mitotic protein monoclonal antibody 2. Additionally, p53 and GADD45 levels were induced in FdUrd-treated HCT116 3-6 cells. DNA double-strand break (DSB) formation was 2-fold higher in MMR-proficient HCT116 3-6 cells after FdUrd treatment, as determined by pulsed-field gel electrophoresis. The formation of DSBs was not the result of enhanced apoptosis in MMR-proficient cells. FdUrd-mediated cytotoxicity was caused by DNA-directed and not RNA-directed effects, because administration of excess thymidine (and not uridine) prevented cytotoxicity, cell cycle arrest, and DSB formation. hMLH1-dependent responses to fluoropyrimidine treatment, which may involve the action of p53 and the formation of DSBs, clearly have clinical relevance for the use of this class of drugs in the treatment of tumors with MMR deficiencies.

Radioimmunoguided imaging of prostate cancer foci with histopathological correlation.

PURPOSE: We have previously presented a technique that fuses ProstaScint and pelvic CT images for the purpose of designing brachytherapy that targets areas at high risk for treatment failure. We now correlate areas of increased intensity seen on ProstaScint-CT fusion images to biopsy results in a series of 7 patients to evaluate the accuracy of this technique in localizing intraprostatic disease. METHODS AND MATERIALS: The 7 patients included in this study were evaluated between June 1998 and March 29, 1999 at Metrohealth Medical Center and University Hospitals of Cleveland in Cleveland, Ohio. ProstaScint and CT scans of each patient were obtained before transperineal biopsy and seed implantation. Each patient's prostate gland was biopsied at 12 separate sites determined independently of Prostascint-CT scan results. RESULTS: When correlated with biopsy results, our method yielded an overall accuracy of 80%: with a sensitivity of 79%, a specificity of 80%, a positive predictive value of 68%, and a negative predictive value of 88%. CONCLUSION: The image fusion of the pelvic CT scan and ProstaScint scan helped identify foci of adenocarcinoma within the prostate that correlated well with biopsy results. These data may be useful to escalate doses in regions containing tumor by either high-dose rate or low-dose rate brachytherapy, as well as by external beam techniques such as intensity modulated radiotherapy (IMRT).

Alternative techniques of intraoperative radiotherapy.

AIMS: The article reviews current techniques for the delivery of intraoperative radiotherapy (IORT). METHODS: Various techniques have been developed to allow for IORT to be given as an adjuvant to surgical resection in the operating room during surgery. This article reviews these different techniques including: dedicated IORT units, intraoperative high dose rate brachytherapy and mobile IORT units such as the Mobetron. RESULTS: IORT may be safely delivered by various methods during operation. Mobile linear accelerators, such as the Mobetron, may allow wider applications of IORT. Preliminary results of IORT at various institutions show promising results. CONCLUSIONS: Widespread applications for IORT are feasible due to improvements in the technology. Future studies should define the proper role for IORT at various disease sites.

Selective radiosensitization of drug-resistant MutS homologue-2 (MSH2) mismatch repair-deficient cells by halogenated thymidine (dThd) analogues: Msh2 mediates dThd analogue DNA levels and the differential cytotoxicity and cell cycle effects of the dThd analogues and 6-thioguanine.

Mismatch repair (MMR) deficiency, which underlies hereditary nonpolyposis colorectal cancer, has recently been linked to a number of sporadic human cancers as well. Deficiency in this repair process renders cells resistant to many clinically active chemotherapy agents. As a result, it is of relevance to find an agent that selectively targets MMR-deficient cells. We have recently shown that the halogenated thymidine (dThd) analogues iododeoxyuridine (IdUrd) and bromodeoxyuridine (BrdUrd) selectively target MutL homologue-1 (MLH1)-deficient human cancer cells for radiosensitization. The levels of IdUrd and BrdUrd in cellular DNA directly correlate with the ability of these analogues to increase the sensitivity of cells and tissues to ionizing radiation, and data from our laboratory have demonstrated that MLH1-mediated MMR status impacts dThd analogue DNA levels, and consequently, analogue-induced radiosensitization. Here, we have extended these studies and show that, both in human and murine cells, MutS homologue-2 (MSH2) is also involved in processing dThd analogues in DNA. Using both E1A-transformed Msh2+/+ and Msh2-/- murine embryonic stem (ES)-derived cells (throughout this report we use Msh2+/+ and Msh2-/- to refer to murine ES-derived cell lines that are wild type or mutant, respectively, for the murine Msh2 gene) and human endometrial cancer cells differing in MSH2 status, we see the classic cytotoxic response to 6-thioguanine (6-TG) in Msh2+/+ and human HEC59/2-4 (MSH2+) MMR-proficient cells, whereas Msh2-/- cells and human HEC59 (MSH2-/-) cells are tolerant (2-log difference) to this agent. In contrast, there is very little cytotoxicity in Msh2+/+ ES-derived and HEC59/2-4 cells to IdUrd, whereas Msh2-/- and HEC59 cells are more sensitive to IdUrd. High-performance liquid chromatography analysis of IdUrd and BrdUrd levels in DNA suggests that this differential cytotoxicity may be due to lower analogue levels in MSH2+ murine and human tumor cells. The DNA levels of IdUrd and BrdUrd continue to decrease over time in Msh2+/+ cells following incubation in drug-free medium, whereas they remain high in Msh2-/- cells. This trend was also found in MSH2-deficient human endometrial cancer cells (HEC59) when compared with HEC59/2-4 (hMsh2-corrected) cells. As a result of higher analogue levels in DNA, Msh2-/- cells are selectively targeted for radiosensitization by IdUrd. Fluorescence-activated cell-sorting analysis of Msh2+/+ and Msh2-/- cells shows that selective toxicity of the halogenated nucleotide analogues is not correlated with a G2-M cell cycle arrest and apoptosis, as is found for selective killing of Msh2+/+ cells by 6-TG. Together, these data demonstrate MSH2 involvement in the processing of IdUrd and BrdUrd in DNA, as well as the differential cytotoxicity and cell cycle effects of the halogenated dThd analogues compared with 6-TG. Therefore, IdUrd and BrdUrd may be used clinically to selectively target both MLH1- and MSH2-deficient, drug-resistant cells for radiosensitization.

Feasibility and acute toxicities of radioimmunoguided prostate brachytherapy.

PURPOSE: We present a technique that fuses pelvic CT scans and ProstaScint images to localize areas of disease within the prostate gland to customize prostate implants. Additionally, the acute toxicity results from the first 43 patients treated with this technique are reviewed. METHODS AND MATERIALS: Between 2/97 and 8/98, 43 patients with clinical stage II prostate adenocarcinoma received ultrasound-guided transperineal implantation of I-125 or Pd-103 seeds. The median patient age was 70 years (range 49-79). Prior to treatment, the median Gleason score and prostate-specific antigen (PSA) were 6 (range 3-8) and 7.5 (range 1.8-16.6 ng/mL), respectively. The median follow-up was 10 months (range 2.9-20.4 months). RESULTS: The median PSA value at 10 months is 0.7 ng/mL. Significant acute complications within the first month following implantation included 13 Grade I urinary symptoms, 24 Grade II urinary symptoms, 6 Grade III symptoms, and no Grade IV complications. Beyond 4 months, complications included 12 Grade I urinary symptoms, 17 Grade II urinary symptoms, 1 Grade III, and 1 Grade IV complications. CONCLUSIONS: The image fusion of the pelvic CT scan and ProstaScint scans helped identify regions within the prostate at high risk of local failure, which were targeted with additional seeds during implantation.

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.

Photodynamic therapy: a new concept in medical treatment.

A new concept in the therapy of both neoplastic and non-neoplastic diseases is discussed in this article. Photodynamic therapy (PDT) involves light activation, in the presence of molecular oxygen, of certain dyes that are taken up by the target tissue. These dyes are termed photosensitizers. The mechanism of interaction of the photosensitizers and light is discussed, along with the effects produced in the target tissue. The present status of clinical PDT is discussed along with the newer photosensitizers being used and their clinical roles. Despite the promising results from earlier clinical trials of PDT, considerable additional work is needed to bring this new modality of treatment into modern clinical practice. Improvements in the area of light source delivery, light dosimetry and the computation of models of treatment are necessary to standardize treatments and ensure proper treatment delivery. Finally, quality assurance issues in the treatment process should be introduced.

Preclinical study of the systemic toxicity and pharmacokinetics of 5-iodo-2-deoxypyrimidinone-2'-deoxyribose as a radiosensitizing prodrug in two, non-rodent animal species: implications for phase I study design.

We have demonstrated previously an improved therapeutic index for oral 5-iodo-2-deoxypyrimidinone-2'-deoxyribose (IPdR) compared with oral and continuous infusion of 5-iodo-2'-deoxyuridine (IUdR) as a radiosensitizing agent using three different human tumor xenografts in athymic mice. IPdR is a prodrug that is efficiently converted to IUdR by a hepatic aldehyde oxidase, resulting in high IPdR and IUdR plasma levels in mice for > or =1 h after p.o. IPdR. Athymic mice tolerated oral IPdR at up to 1500 mg/kg/day given four times per day for 6-14 days without significant systemic toxicities. In anticipation of an investigational new drug application for the first clinical Phase I and pharmacology study of oral IPdR in humans, we studied the drug pharmacokinetics and host toxicities in two non-rodent, animal species. For the IPdR systemic toxicity and toxicology study, twenty-four male or female ferrets were randomly assigned to four IPdR dosage groups receiving 0, 15, 150, and 1500 mg/kg/day by oral gavage x 14 days prior to sacrifice on study day 15. All ferrets survived the 14-day treatment. Ferrets receiving 1500 mg/kg/day showed observable systemic toxicities with diarrhea, emesis, weight loss, and decreased motor activity beginning at days 5-8 of the 14-day schedule. Overall, both male and female ferrets receiving IPdR at 1500 mg/kg/day experienced significant weight loss (9 and 19%, respectively) compared with controls after the 14-day treatment. No weight loss or other systemic toxicities were observed in other IPdR dosage groups. Grossly, no anatomical lesions were noted at complete necropsy, although liver weights were increased in both male and female ferrets in the two higher IPdR dosage groups. Histologically, IPdR-treated animals showed dose-dependent microscopic changes in liver consisting of minimal to moderate cytoplasmic vacuolation of hepatocytes, which either occurred in the periportal area (high dosage group) or diffusely throughout the liver (lower dosage groups). Female ferrets in the highest IPdR dose group also showed decreased kidney and uterus weights at autopsy without any associated histological changes. No histological changes were found in central nervous system tissues. No significant abnormalities in blood cell counts, liver function tests, kidney function tests, or urinalysis were noted. Hepatic aldehyde oxidase activity was decreased to approximately 50 and 30% of control ferrets in the two higher IPdR dosage groups, respectively, after the 14-day treatment period. The % IUdR-DNA incorporation in ferret bone marrow at the completion of IPdR treatment was < or =0.05% in the two lower dosage groups and approximately 2% in the 1500 mg/kg/day dosage group. The % IUdR-DNA in normal liver was < or =0.05% in all IPdR dosage groups. In a pharmacokinetic study in four Rhesus monkeys, we determined the plasma concentrations of IPdR after a single i.v. bolus of 50 mg/kg over 20 min. Using a two-compartment model to fit the plasma pharmacokinetic data, we found that IPdR was cleared in these non-human primates in a biexponential manner with an initial rapid distributive phase (mean T1/2alpha = 6.5 min), followed by an elimination phase with a mean T1/2 of 63 min. The mean maximum plasma concentration of IPdR was 124+/-43 microM with a mean total body clearance of 1.75+/-0.95 l/h/kg. IPdR was below detection (<0.5 microM) in the cerebrospinal fluid. We conclude that there are dose-limiting systemic toxicities to a 14-day schedule of p.o. IPdR at 1500 mg/kg/day in ferrets that were not found previously in athymic mice. However, no significant hematological, biochemical, or histopathological changes were found. Hepatic aldehyde oxidase activity was reduced in a dose-dependent in ferret liver, suggesting partial enzyme saturation by this IPdR schedule. The plasma pharmacokinetic profile in Rhesus monkeys showing biexponential clearance is similar to our published data in athymic mice. These data are being applied

Nuclear clusterin/XIP8, an x-ray-induced Ku70-binding protein that signals cell death.

Clusterin [CLU, a.k.a. TRPM-2, SGP-2, or ionizing radiation (IR)-induced protein-8 (XIP8)] was implicated in apoptosis, tissue injury, and aging. Its function remains elusive. We reisolated CLU/XIP8 by yeast two-hybrid analyses using as bait the DNA double-strand break repair protein Ku70. We show that a delayed (2-3 days), low-dose (0.02-10 Gy) IR-inducible nuclear CLU/XIP8 protein coimmunoprecipitated and colocalized (by confocal microscopy) in vivo with Ku70/Ku80, a DNA damage sensor and key double-strand break repair protein, in human MCF-7:WS8 breast cancer cells. Overexpression of nuclear CLU/XIP8 or its minimal Ku70 binding domain (120 aa of CLU/XIP8 C terminus) in nonirradiated MCF-7:WS8 cells dramatically reduced cell growth and colony-forming ability concomitant with increased G(1) cell cycle checkpoint arrest and increased cell death. Enhanced expression and accumulation of nuclear CLU/XIP8-Ku70/Ku80 complexes appears to be an important cell death signal after IR exposure.

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.

Radiosensitivity of thymidylate synthase-deficient human tumor cells is affected by progression through the G1 restriction point into S-phase: implications for fluoropyrimidine radiosensitization.

Recent studies of fluoropyrimidine (FP)-mediated radiosensitization (RS) have focused on the molecular mechanisms underlying regulation of the cell cycle, particularly at the G1-S transition. Although thymidylate synthase (TS) inhibition by FP is necessary, we hypothesize that FP-RS is temporally dependent on progression of cells into S-phase under conditions of altered deoxynucleotide triphosphate pools, particularly an increased dATP:dTTP ratio, which subsequently results in enhanced DNA fragmentation and cell death. To better understand the mechanism of FP-RS, we characterized the cellular and biochemical responses to ionizing radiation (IR) alone, using different synchronization techniques in two isogenic, TS-deficient mutant cell lines, JH-1 (TS-) and JH-2 (Thy4), derived previously from a human colon cancer cell line. After G0 synchronization by leucine deprivation, these clones differ under subsequent growth conditions and dThd withdrawal: JH-2 cells have an intact G1 arrest (>72 h) and delayed cell death (>96 h), whereas JH-1 cells progress rapidly into early S-phase and undergo acute cell death (<24 h). No difference in the late S-phase and G2-M cell populations were noted between these growth-stimulated, G0-synchronized TS-deficient cell lines with dThd withdrawal. Biochemically, the intracellular ratio of dATP:dTTP increased substantially in JH-1 cells as cells progressed into early S-phase compared with JH-2 cells, which remained in G1 phase. Synchronized JH-1 cells showed significantly decreased clonogenic survival and an increase in DNA fragmentation after IR when compared with JH-2 cells. RS was demonstrated by an increase in alpha and decrease in beta, using linear quadratic analyses. An alternative synchronization technique used mimosine to induce a block in late G1, close to G1-S border. Both JH-1 and JH-2 cells, synchronized in late G1 and following growth stimulation, now progressed into S-phase identically (<24 h), with similarly increased dATP:dTTP ratios under dThd withdrawal conditions. These late G1-synchronized JH-1 and JH-2 cells also showed a comparable reduction in clonogenic survival and similar patterns of increased DNA fragmentation following IR. We suggest, based on the cellular and biochemical differences in response to IR between G0- and late G1-synchronized cells, that S-phase progression through the G1 restriction point under an altered (increased) dATP:dTTP ratio is a major determinant of FP-RS.