High dose-rate brachytherapy of prostate cancer utilising Iridium-192 after-loading technique: technical and methodological aspects.

The aim of this study was to focus on certain characteristic problems associated with Iridium-192 high dose-rate brachytherapy (Ir-192 HDR-BT) in combination with external beam radiation therapy (EBRT) in the treatment of patients with localised prostate cancer. Over a period of 16 years, >2,000 patients with prostate cancer have been treated in Sweden with a combination of two fractions of 10 Gy Ir-192 HDR-BT and 50 Gy of fractionated EBRT. Although this treatment is usually well tolerated, there are biological and technical factors to be considered before and during the treatment of the patient to avoid side effects or under-treatment of the target volume. Some of the problems facing the doctors are transducer stability, needle deviation, target definition, target motion, pubic arch interference, concomitant diseases and tolerance doses for different organs at risk. These problems are discussed and possible solutions are presented in this study.

Effect of D,L-buthionine-S,R-sulfoximine on cytotoxicity and DNA cross-linking induced by bifunctional DNA-reactive cytostatic drugs in human melanoma cells.

The effects of D,L-buthionine-S,R-sulfoximine (BSO) on cytotoxicity and DNA cross-linking induced by bifunctional DNA-reactive cytostatic agents in a human melanoma cell line (RPMI 8322) were investigated. RPMI 8322 cells were exposed to 0.01 mM BSO for 24 h, which resulted in a decrease in cellular glutathione to 14% without any reduction of cell proliferation or plating efficiency. BSO pretreatment significantly enhanced cytotoxicity of melphalan with a dose modification factor (DMF) of 3.4 and nitrogen mustard (HN2) (DMF 3.3). The increased cytotoxicity was paralleled by similar increases in DNA cross-linking (melphalan: DMF 2.2, HN2: DNF 2.5). A small but significant potentiation by BSO of cis-diamminedichloroplatinum(II) toxicity was seen (DMF 1.5), with a corresponding minor but significant increase in DNA cross-linking (DMF 1.1). Similarly, the potentiation of bis-chloroethylnitrosurea toxicity was small but significant (DMF 1.1), with no significant increase in DNA cross-linking (DMF 1.0). No effect of BSO pretreatment on the rate of removal of HN2-induced DNA cross-links was observed. Thus, the observed sensitization of RPMI 8322 cells to melphalan, HN2, cis-diamminedichloroplatinum(II), and bis-chloroethylnitrosourea was correlated to similar changes in drug-induced DNA cross-linking. Despite the increased cytotoxicity and DNA cross-linking BSO did not significantly increase the intracellular concentration of intact melphalan. These findings support the hypothesis that the potentiation of the cytotoxicity of bifunctional alkylating agents by BSO is due to an increased DNA cross-linking caused by a reduced intracellular conjugation of drug with glutathione, which results in an increased binding of drug to DNA targets.

Dose and time dependent apoptotic response in a human melanoma cell line exposed to accelerated boron ions at four different LET.

The aim was to investigate and compare the influence of linear energy transfer (LET), dose and time on the induction of apoptosis in a human melanoma cell line exposed to accelerated light boron ((10)B) ions and photons. Cells were exposed in vitro to doses up to 6 Gy accelerated boron ions (40, 80, 125 and 160 eV nm(-1)) and up to 12 Gy photons (0.2 eV nm(-1)). The induction of apoptosis was measured up to 9 days after irradiation using morphological characterization of apoptotic cells and bodies. In parallel, measurements of cell-cycle distribution, monitored by DNA flow cytometry, and cell survival based on the clonogenic cell survival assay, were performed. In addition, the induction and repair of DNA double-strand breaks (DSB), using pulsed-field gel electrophoresis (PFGE) were studied. Accelerated boron ions induced a significant increase in apoptosis as compared with photons at all time points studied. At 1-5 h the percentage of radiation-induced apoptotic cells increased with both dose and LET. At the later time points (24-216 h) the apoptotic response was more complex and did not increase in a strictly LET-dependent manner. The early premitotic apoptotic cells disappeared at 24 h following exposure to the highest LET (160 eV nm(-1)). A postmitotic apoptotic response was seen after release of the dose-, time- and LET-dependent G2/M accumulations. The loss of clonogenic ability was dose- and LET-dependent and the fraction of un-rejoined DSB increased with increasing LET. Despite the LET-dependent clonogenic cell killing, it was not possible to measure quantitatively a LET-dependent apoptotic response. This was due to the different time course of appearance and disappearance of apoptotic cells.

Comparative study of two human melanoma cell lines with different sensitivities to mustine and cisplatin.

The cytotoxic effects of the bifunctional DNA-reactive drugs cisplatin, mustine and melphalan were studied in two human melanoma cell lines, RPMI 8322 and A 375. A 375 cells were 3.0 times more sensitive to cisplatin and 1.5 times more sensitive to mustine than RPMI 8322 cells. In contrast, A 375 cells were less sensitive to melphalan than RPMI 8322 cells. The increased sensitivity of A 375 cells was parallelled by an increased induction of DNA interstrand crosslinks following exposure to cisplatin and mustine. After cisplatin exposure A 375 cells also showed higher levels of platinum-DNA intrastrand adducts than RPMI 8322 cells. The increased effect of cisplatin in A 375 cells was not due to an increased drug accumulation in these cells. The higher sensitivity of A 375 cells to cisplatin may be related to lower intranuclear levels of glutathione, compared to RPMI 8322 cell nuclei, while the sensitivity to mustine may depend on lower overall levels of glutathione than in RPMI 8322 cells.

Role of O6-methylguanine-DNA methyltransferase, glutathione transferase M3-3 and glutathione in resistance to carmustine in a human non-small cell lung cancer cell line.

O6-methylguanine-DNA methyltransferase (MGMT), glutathione transferase (GST) M3-3 and glutathione (GSH) have all been implicated in the resistance of cells to the cytostatic drug carmustine. U1810, a human non-small cell lung cancer cell line, expresses all of these putative resistance factors. The U1810 cells show a 4.4-fold lower sensitivity to carmustine compared with the U1690 cell line, a human small cell lung cancer cell line lacking detectable levels of both MGMT and GST M3-3. We investigated the effect of the MGMT inhibitor O6-benzylguanine, the GST inhibitor ethacrynic acid and the GSH synthesis inhibitor D,L-buthionine-S,R-sulfoximine (BSO) on the cytotoxicity of carmustine to U1810 cells. No potentiation to carmustine was observed after treatment with ethacrynic acid, while a 2-fold potentiation was found after exposure to O6-benzylguanine. Depletion of GSH with BSO showed a similar sensitising effect as that obtained with O6-benzylguanine. Thus, MGMT and GSH are the predominant resistance factors to carmustine in the U1810 cell line, whereas it is unclear whether GST M3-3 plays any role.

Specificity of endogenous glutathione in determining the oxygen enhancement of cellular radiosensitivity.

Oxygen enhancement ratios (OER) determined with single-strand DNA breaks as end-point of radiation effect were lower for cells with genetically determined glutathione (GSH) deficiency, than for cells depleted of GSH artificially to a corresponding degree by treatment with buthionine sulfoximine (BSO). In view of a considerable non-protein thiol (NPSH) accumulation found in both cases, the difference can be attributed to different NPSH components. Those in the BSO-treated cells having the capacity to substitute for GSH in the processes which define OER. As an alternative explanation, the particular location of GSH in GSH-proficient cells in close contact with the critical radiation target can be considered. At these sites, as indicated by measurements of NPSH in isolated nuclei, BSO may not deplete thiols as efficiently as at other sites. Observations on a persisting difference found between the OER of genetically GSH-deficient and proficient cells after treatment with dithiothreitol (DTT) can also be given a spatial explanation, assuming that exogenous thiols may not reach the critical sites in the nucleus in efficient concentrations.

Selective toxicity of buthionine sulfoximine (BSO) to melanoma cells in vitro and in vivo.

PURPOSE: Glutathione (GSH) was found to occur in relatively high concentrations in melanoma cells. The purpose of this study was to test the possible cytotoxic effects of an artificial decrease of the elevated GSH level. METHODS AND MATERIALS: The tests were made in vitro and in vivo. In the former case, a total of 11 rodent and human cell lines were studied of which seven were derived from melanomas. After treatment with buthionine sulfoximine (BSO), the decrease of GSH content of the cells and their clonogenic survival was determined. In the in vivo system, single cell suspensions of a subline of the B16 mouse melanoma were injected intravenously into immunocompetent and preirradiated recipients which were subsequently treated with BSO intraperitoneally. Survival time, formation of lung colonies and the weight of metastatic tumor mass in the lungs were the criteria of the BSO effect on the tumor cells. RESULTS: The decrease of the GSH level by BSO was associated with impaired clonogenic survival of the melanoma cells in vitro. Nonmelanoma cells were less affected. BSO treatment of mice inoculated intravenously with melanoma cells resulted in prolonged survival of the animals and impaired metastatic spread of the tumor cells. CONCLUSION: Melanoma cells are particularly sensitive to disturbance of GSH metabolism by treatment with BSO. In view of this selective cytotoxicity of BSO, treatment with this substance may afford a promising therapeutic potential for melanoma.

Radiation-dose dependence of the enhancement of DNA-damage in cells treated with the hypoxic radiosensitizer ak-2123, a 3-nitrotriazole derivative.

The radiosensitizing effect of AK-2123, a 3-nitrotriazole, and of misonidazole, a 2-nitroimidazole, was studied on hypoxically irradiated Chinese hamster cells (V79-379A) with DNA damage as end-point. The damage was estimated with the alkaline unwinding assay and the DNA precipitation technique, carried out in parallel or in separate experimental series. The sensitizers were used in different concentrations together with standard radiation doses of 30, 60 and 90 Gy. Sensitizer-untreated cell cultures served as controls. The data indicated a radiation dose-dependent sensitization when the effect of AK-2123 was tested with the two assays, the lower radiation dose being associated with larger sensitization. In contrast, sensitization was found to be dose-independent when the effect of misonidazole was tested.

Adrenocortical carcinoma - a clinical and immunohistochemical study.

Twenty-one patients with adrenocortical carcinoma (ACC) were investigated. No correlation was found between size of the primary tumour and the occurrence of metastases, and no correlation was observed between tumour diameter and the functional status of the tumour or survival. Cytokeratins (CK) were expressed in 31% and vimentin (VIM) in 41% of cases. Epithelial membrane antigen (EMA) was detected in one patient with a non-functioning tumour, whereas carcinoembryonic antigen (CEA) was absent in all rumours. The antigens investigated showed no correlation with the morphology or functional status of the tumour. The role of immunohistochemical characterization of this heterogeneous malignancy is discussed.

Radiosensitization with a 3-nitrotriazole (ak-2123).

We investigated to clarify the mechanism of action of AK-2123. V79 cells were used, and the effect of the substance was estimated with different end-points viz. induction of DNA breaks, micronuclei and clonogenic survival after hypoxic irradiation with different X-ray doses. After treatment with 0.1 mmol dm(-3) of the substance, sensitization by a factor of 1.8, 1.1 and 1.4 respectively, was calculated. By determining the enhancement ratio after treatment with the drug at 7-8 different concentrations varying between 0.001 and 50 mmol dm(-3), the concentration of the substance for half of the maximum sensitization (K-value) was estimated to be 40 mu mol dm(-3) (DNA breaks), 1200 mu mol dm(-3) (micronuclei) and 2000 mu mol dm(-3) (survival); the maximum sensitizer enhancement ratio was 2.4 (DNA breaks), 2.3 (micronuclei) and 2.4 (survival). No cytotoxic effects were found when the cells were treated with the drug in different concentrations for 30 minutes. However, after longer periods the compound was found to be cytotoxic both under aerobic and hypoxic conditions, in a concentration and time dependent manner. A post-irradiation enhancement of the sensitivity was noted when the drug was left in the medium for 24 hours after hypoxic as well as aerobic radiation exposure.

The effect of modulation of glutathione cellular content on busulphan-induced cytotoxicity on hematopoietic cells in vitro and in vivo.

Busulphan is used in conditioning regimens prior to SCT. A relationship between exposure to busulphan, expressed as an area under the plasma concentration time curve (AUC), and effect and/or adverse effects, such as veno-occlusive disease (VOD), was reported. Exhaustion of glutathione (GSH) contributes to VOD and modulation of intracellular levels of GSH influences bulsulphan-induced toxicity in hepatocytes. Thus, increase of GSH might serve as prophylaxis against VOD. However, it should not interfere with the myeloablative effects of busulphan. We investigated the relationship between exposure to busulphan, and its in vitro toxicity to CD34(+) hematopoietic progenitors from volunteers using clonogenic assays. Busulphan inhibited colony formation by CD34(+) cells in an AUC-dependent manner. Myeloid progenitors were more sensitive than erythroid progenitors, expressed as 100% inhibition of colony formation (68.7 +/- 7.5 microg.h/ml and 140.3 +/- 35.7, respectively). The observed exposure corresponds to the total AUC obtained in patients treated with busulphan (1 mg/kg/day) for 4 days. Secondly, we studied the effect of modulation of GSH cellular levels on busulphan-induced toxicity in vitro in CD34(+) cells from volunteers, and in vivo in bone marrow cells from Balb/c mice. The intracellular concentration of GSH was increased or decreased by treatment with N-acetylcysteine or buthionine sulfoximine, respectively. Neither in vitro nor in vivo treatment with GSH modulators affected the hematological toxicity of busulphan. Thus, N-acetylcysteine would not interfere with the myeloablative effect of busulphan and therefore it is a potential candidate for VOD prophylaxis during busulphan-based conditioning regimens.

Repairable-conditionally repairable damage model based on dual Poisson processes.

The advent of intensity-modulated radiation therapy makes it increasingly important to model the response accurately when large volumes of normal tissues are irradiated by controlled graded dose distributions aimed at maximizing tumor cure and minimizing normal tissue toxicity. The cell survival model proposed here is very useful and flexible for accurate description of the response of healthy tissues as well as tumors in classical and truly radiobiologically optimized radiation therapy. The repairable-conditionally repairable (RCR) model distinguishes between two different types of damage, namely the potentially repairable, which may also be lethal, i.e. if unrepaired or misrepaired, and the conditionally repairable, which may be repaired or may lead to apoptosis if it has not been repaired correctly. When potentially repairable damage is being repaired, for example by nonhomologous end joining, conditionally repairable damage may require in addition a high-fidelity correction by homologous repair. The induction of both types of damage is assumed to be described by Poisson statistics. The resultant cell survival expression has the unique ability to fit most experimental data well at low doses (the initial hypersensitive range), intermediate doses (on the shoulder of the survival curve), and high doses (on the quasi-exponential region of the survival curve). The complete Poisson expression can be approximated well by a simple bi-exponential cell survival expression, S(D) = e(-aD) + bDe(-cD), where the first term describes the survival of undamaged cells and the last term represents survival after complete repair of sublethal damage. The bi-exponential expression makes it easy to derive D(0), D(q), n and alpha, beta values to facilitate comparison with classical cell survival models.

A comparison of positron emission tomography (pet) utilizing C-11 methionine and computerized-tomography in the diagnosis of renal-cell adenocarcinoma.

Positron emission tomography (PET) is a radionuclide method that quantifies physiological and metabolic parameters in vivo. PET with L-methyl-C-11-methionine as a tracer was performed in 13 patients with advanced renal cell carcinoma and twenty-two lesions were evaluated. Two of 13 patients still had their primary renal tumours. The PET images of all the patients showed a good correlation with the computerized tomography (CT) images, and detected not only the primary tumour but also metastases. PET utilizing 11-methionine was efficient in visualizing metastatic lesions in the thorax. The conclusion of this pilot study is that the PET technique with C-11-methionine as a tracer can be used in the diagnosis of advanced renal cell adenocarcinoma to visualize and characterize the primary renal tumour and its metastases in vivo.

Positron emission tomography utilizing C-11 5-hydroxytryptophan, plasma biochemistry and neuroendocrine immunohistochemistry of metastatic renal-cell carcinoma.

The aim of this study was to characterise metastatic renal cell carcinoma in 18 patients with positron emission tomography (PET) utilising C-11-5-hydroxytryptophan, plasma biochemistry and neuroendocrine immunochemistry. Of these 18 patients, ten underwent the PET investigations. The standardised uptake values (SUVs) in hepatic deposits were higher than those in pulmonary lesions, with mean values of 3.15 and 2.35, respectively. The immunohistochemical study included staining of 10/18 surgical tumour specimens with antibodies reactive with chromogranin (Cg), neurone-specific enolase (NSE), and synaptophysin (Sy). In 17/18 patients, plasma measurements of the neuroendocrine peptides, CgA, CgB, pancreastatin, and serotonin, were performed. The results obtained in this study show that PET with C-11-5-hydroxytryptophan, a precursor in serotonin synthesis in neuroendocrine cells, can be utilised to visualise primary renal cell cancer and its secondaries in vivo. The results obtained also suggest that neuroendocrine differentiation may occur in human RCC.

Estramustine a radio sensitising agent.

The present study revealed that estramustine acts as a radio sensitising agent on the human renal cell cancer cell lines, A498 and CAKI-2. In vitro experiments used the Bürker chamber technique. Both cell lines were markedly resistant to external beam irradiation. While pretreatment of the cell cultures with estramustine prior to external beam irradiation revealed an arrest of cell growth in both cell lines. The results of this study suggest that estramustine could be utilised as a radiosensitizing agent. This in turn could open a new method for the management of patients with advanced renal cell carcinoma (RCC).

Serum concentrations of VEGF and b-FGF in renal cell, prostate and urinary bladder carcinomas.

Sixty nine patients with urogenital cancers (renal, bladder and prostate cancer) were studied to determine whether the serum concentrations of Vascular Endothelial Growth Factor (VEGF) and basic Fibroblast Growth Factor (b-FGF) reflected the status of the patients and/or the prognosis of the disease. Of the patients included in this study, renal cell carcinoma patients expressed the highest levels of VEGF indicating that these tumours are more VEGF dependent. The values of b-FGF could be considered normal in all three malignancies. No correlation was observed between the expression of VEGF and b-FGF, nor between VEGF and b-FGF and patients survival.

Postoperative radiotherapy after prostatectomy can be associated with severe side effects.

This retrospective study was initiated to evaluate the efficacy and side effects of post-prostatectomy external beam radiation therapy (XRT) with a short time interval between surgery and irradiation in patients with prostate adenocarcinoma. Sixteen patients were investigated. The overall results in this study were 3 deaths due to recurring disease and two relapses after an average follow-up of 60 months. Severe side effects were observed. Two patients required surgical intervention due to severe post-radiotherapy side effects. The reason for this could be the high dose delivered to peripheral organs and/or a too short time interval between surgery and postoperative XRT. The results of this study confirmed that postoperative XRT can improve local control frequency in prostate carcinomas. It is recommended that the time interval between surgery and postoperative radiotherapy should to be 3-6 month.

Angiogenic factors: vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF) are not necessarily elevated in patients with advanced renal cell carcinoma.

Serum analysis of Vascular Endothelial Growth Factor (VEGF) and basic Fibroblast Growth Factor (b-FGF) levels were studied in 53 patients with renal cell carcinoma (RCC). Approximately 2/3 of the patients had disseminated disease at diagnosis, the remainder had no evidence of metastases. The results confirmed that VEGF has a major role in the angiogenesis of RCC. No correlation was observed between VEGF and/or b-FGF and the presence or absence of metastases, nor was any correlation observed between VEGF and/or b-FGF and patient survival. Thus, to utilise VEGF and/or b-FGF as a tumour marker at the time of diagnosis to predict patients with a high risk of progression, where an adjuvant therapeutic approach would be of great value, seems to be limited. Not all patients with RCC exhibited elevated serum levels of VEGF and/or b-FGF. No correlation was observed between tumour stage and serum levels of these angiogenic peptides.

Neuroendocrine markers; chromogranin, pancreastatin and serotonin in the management of patients with advanced renal cell carcinoma.

A fraction of patients with renal cell carcinoma (RCC) exhibit elevated plasma levels of neuroendocrine (NE) markers, including chromogranin (Cg) A and B, pancreastatin and serotonin. This may suggest neuroendocrine involvement in human RCC. Twenty eight patients (24 men and 4 women) with advanced RCC were included in this study. The ongoing therapy was either tamoxifen, or interleukin-2 (IL-2); alpha interferon and tamoxifen. Plasma analyses of NE markers revealed elevated levels of CgA and serotonin in 28/28 (100%) patients. CgB was elevated in 5/16 (31%) patients, whereas no elevation of pancreastatin was observed in these 16/28 patients. Although the plasma levels of NE markers did not statistically or significantly influence the prognosis of the disease it was observed that patients with elevated NE markers had a less aggressive disease and lived longer than patients with normal or only slightly elevated NE markers. Immunohistochemical analyses of tumour specimens from 10 patients were chromogranin, serotonin and synaptophysin negative, but all were neurone-specific enolase (NSE) positive. The results obtained are of interest. However, extended studies are needed to define the role of these NE markers in the clinical management of patients with RCC.

Melphalan-induced DNA cross-linking in human melanoma cells and phytohaemagglutinin-stimulated lymphocytes in relation to intracellular drug content and cellular levels of glutathione.

Melphalan-induced DNA cross-linking was compared in a human melanoma cell line (RPMI 8322) and in phytohaemagglutinin (PHA)-stimulated lymphocytes. In both cell types a delayed induction of DNA cross-links was observed, with maximum DNA cross-linking occurring 6-12 hours after drug exposure. Significantly higher peak levels of DNA cross-links were found in PHA-stimulated lymphocytes, total DNA cross-linking being 2.5 times and DNA interstrand cross-linking 2.2 times higher. The intracellular content of free melphalan was 1.3-fold higher in RPMI 8322 cells, thus the lower DNA cross-linking was not due to a lower drug concentration in these cells. RPMI 8322 cells had a 1.8-fold higher level of glutathione, possibly indicating a higher capacity of these cells to inactivate melphalan.