Biomedical subjects

A C Begg

Publications and source records attributed to A C Begg.

At least 55 records · Page 3Linked to original sources

PubMed1994

Differential repair of radiation-induced DNA damage in cells of human squamous cell carcinoma and the effect of caffeine and cysteamine on induction and repair of DNA double-strand breaks.

The goal of these experiments was to investigate further the relationship between DNA double-strand breaks and cell killing in human tumor cells, first by comparing different cell lines, and second by radiomodification studies. Field-inversion gel electrophoresis was used to quantify double-strand breaks. Two subclones of the radioresistant human squamous cell carcinoma line SQ20B (SQD9 and SQG6) were compared. These subclones differed in DNA index by a factor of 1.7 but showed the same resistance to radiation as cells of the parental cell line. It was found that, although induction of DSBs was not significantly different in the two cell lines, the t1/2 of the fast component of repair was significantly shorter for SQD9 cells, leading to greater overall repair which was not reflected in increased survival. Caffeine and cysteamine were tested as modifiers of radiosensitivity, using the radioresistant SQ20B line and the radiosensitive SCC61 cell line. No effect of caffeine was seen when the drug was present only during irradiation. Postirradiation incubations with caffeine, however, resulted in a dose reduction factor greater than 2.0 in cell survival for both cell lines. In contrast, induction of DSBs was reduced by caffeine, and no effect on DSB repair was observed. Cysteamine led to a dose protection factor greater than 1.8 in cell survival in both cell lines. A reduction in induced DSBs was found at high doses corresponding approximately with the increase in cell survival. Over the same (low) dose range, however, the correlation between DSB induction and cell killing was poor. These data indicate that DSB induction does not correlate well with cell killing either for different cell lines, for radiochemical modification (cysteamine) or for some other types of modification (caffeine).

Caffeine↗

PubMed1994

Intrinsic radiosensitivity and chromosome aberration analysis using fluorescence in situ hybridization in cells of two human tumor cell lines.

The survival curves for cells of two human tumor cell lines, HT29 and MeWo, have been defined using a Dynamic Microscopic Imaging Processing Scanner (DMIPS). There are two major differences between these two cell lines: (a) HT29 is more radioresistant than MeWo (surviving fraction at 2 Gy of 74 and 27%, respectively) and (b) HT29 presents a marked multiphasic survival curve with hypersensitivity at low doses (< 0.5 Gy) followed by an increase in radioresistance at higher doses which we have interpreted as "induced radioresistance"; this phenomenon is much less pronounced for the more radiosensitive cell line MeWo. We have now measured in these two cell lines the stable chromosomal aberrations and fragments, with the method of fluorescence in situ hybridization (FISH). We have analyzed chromosome 4, which does not have spontaneous translocations in either of these two cell lines. A dose-effect relationship was studied for radiation doses up to 5 Gy. At all doses, both translocations and breaks are more frequent in the radiosensitive cell line MeWo compared to the radioresistant cell line HT29. The correlation between survival and translocations is different for HT29 and MeWo, thus indicating that another factor(s) may be involved in cell killing in these lines.

Cell Survival↗

PubMed1993

Selectivity of boron carriers for boron neutron capture therapy: pharmacological studies with borocaptate sodium, L-boronophenylalanine and boric acid in murine tumors.

Borocaptate sodium (BSH) and L-boronophenylalanine (L-BPA) are two boron carriers used for boron neutron capture therapy (BNCT) in the treatment of glioblastoma and melanoma, respectively. The suitability of these two compounds was evaluated on the basis of pharmacokinetic studies aiming at characterizing their biodistribution, tumor uptake and tumor selectivity. Boric acid was also used as a reference compound since it is nonselective and relatively freely diffusible. The compounds were investigated in two tumor models, a B16 pigmented melanoma and the RIF1 sarcoma. Mice were sacrificed after different boron doses at various post-injection times and tissue and plasma levels measured using inductively coupled plasma atomic emission spectroscopy (ICP-AES). The proposed minimum effective tumor boron concentration of 15 ppm was achieved in both tumor models for the three compounds tested, although only for L-BPA in the melanoma was this achieved when tumor-plasma ratios were above 1. In the RIF1 model, maximum tumor concentrations of 44 and 31 ppm B were reached after administration of 50 micrograms B/g body weight for boric acid and BSH, respectively. After administration of 12.5 micrograms B/g of L-BPA, maximum concentrations of 15 and 21 ppm were found in the RIF1 and B16 models, respectively. Tumor-plasma ratios (TPR) for BSH remained close to or below unity at all times studied in both tumors. Brain levels of BSH were very low, however, leading to tumor-brain ratios markedly greater than 1 at all times. L-BPA and boric acid showed TPR values above unity in both tumor models, reaching 3.2 in B16.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Cell kinetic analysis of murine squamous cell carcinomas: a comparison of single versus double labelling using flow cytometry and immunohistochemistry.

The study was originally set up to measure accurate cell kinetic parameters in two murine squamous cell carcinomas (scc) for comparison with radiobiological data on proliferation during radiotherapy. The tumours, AT84 and AT478, were both moderately well differentiated aneuploid scc. In the course of the study, several comparisons of techniques were made in two different centres. This paper reports on the results of those comparisons involving two different detection methods (flow cytometry and immunohistochemistry), single vs double labelling, and in vivo and in vitro labelling, the latter using tissue slices incubated under high pressure oxygen. Pulse labelling studies with bromodeoxyuridine (BrdUrd) showed that the labelling indices (LI) were not significantly different after in vitro or in vivo labelling. In addition, the flow cytometry (FCM) and immunohistochemistry (IHC) methods also gave labelling indices which were not significantly different. Only tumour cells were analysed in these studies by selecting cells on the basis of aneuploidy (FCM) or morphology (IHC). The DNA synthesis time of the tumour cells were analysed by both techniques. For FCM, the Relative Movement method was used (Begg et al., 1985). For IHC, a double labelling method was used, employing BrdUrd and triated thymidine (3H-TdR) administered several hours apart, detected simultaneously using immunoperoxidase and autoradiography, respectively. When both labels were administered in vivo, there was good agreement for Ts between the FCM and IHC methods. Attempts were also made to measure Ts in vitro using both techniques. With double labelling, it was found that cells did not take up the second label, implying a failure of cycle progression. This was confirmed by FCM results, showing no movement of labelled cells through the S-phase, despite an initially high uptake. This could not be influenced by lowering the DNA precursor concentration or by adding foetal calf serum. This indicates that DNA synthesis times are difficult or impossible to measure in vitro in fresh tumour explants. Finally, the double labelling IHC method allowed intratumoural variations of both LI and Ts to be studied. Both parameters were found to vary markedly throughout the tumour volume, particularly for larger tumours (600 mg), giving calculated local potential doubling time values (Tpot) ranging from 1-7 days.

Animals↗

PubMed1993

Lack of correlation of human fibroblast radiosensitivity in vitro with early skin reactions in patients undergoing radiotherapy.

Fibroblasts from breast cancer patients were obtained as outgrowths in vitro from punch biopsies and their radiosensitivity tested in early passages. Skin erythema reactions in the same patients were also measured, as degree of redness using reflectance spectrophotometry. Measurements were taken before and during a 4-week radiotherapy treatment with electrons to the thoracic wall. Of 59 biopsies studied, radiosensitivity and erythema were concurrently studied in 32. In 24, evaluable data from both clinic and laboratory were obtained. A population growth assay in 96-well plates, using absorption of sulphur rhodamine B as the stain for cell numbers, showed good agreement with the colony-formation assay. Plating efficiencies and growth rates in the colony assay were higher using human serum in place of foetal calf serum. Cell survival curves with human serum were mostly exponential with little shoulder. The parameters of survival at 2 Gy (SF2) and the dose required to give 10% survival (D10) were used in the correlations with clinical data; these were 0.25 +/- 0.09 and 3.03 +/- 0.50 Gy, respectively. There was a strong correlation between these two survival curve parameters (r = 0.98). Skin redness was found to linearly increase with time during radiotherapy. The slope of the increase differed markedly from patient to patient, with a range of a factor approx. 10. No correlation was found between SF2 and erythema response in the 24 evaluable patients (r = 0.13, p > 0.5). A similar lack of correlation was found using D10 as the radiosensitivity parameter (r = 0.12, p > 0.5). These data indicate that fibroblast radiosensitivity measured in vitro cannot be used to predict erythema reactions to radiotherapy in breast cancer patients.

Breast Neoplasms↗

PubMed1993

Radiation-induced DNA damage and repair in radiosensitive and radioresistant human tumour cells measured by field inversion gel electrophoresis.

Radiation-induced DNA damage induction and repair was measured in two human squamous carcinoma cell lines with differing radiosensitive. Experiments were carried out with field inversion gel electrophoresis (FIGE), adapted to measure DNA double strand break (DSB) induction and repair in unlabelled cells. The sensitivity of the method was increased by introducing a hybridization membrane into the agarose gel. Damaged DNA accumulated on one spot on the membrane resulting in high local concentrations. This DNA was quantified using radioactively-labelled total human DNA as a probe. Dose response experiments for damage induction correlated well with the results using prelabelled cells. Linear DNA damage induction curves were observed with a sensitivity for the post-labelling method of 1 Gy. No differences in DSB induction were found, however, between the radiosensitive SCC61 and the radioresistant SQ20B cell line. Repair experiments were carried out with trypsinized cells with different doses and repair temperatures. The 10, 25 and 50 Gy doses resulted in 6, 13 and 50% of the DNA migrating out of the plug at 0 h. For both the cell lines 75-85% of the initial damage was repaired within 1 h at 37 degrees C at all three radiation doses, i.e. no significant differences were observed in repair rates or extent between the two cell lines. At 24 degrees C repair was slower than at 37 degrees C, and at 0 degree C no repair was observed. In summary, radiosensitivity differences at physiological temperatures could not be explained by differences in either induction or repair of DNA damage as measured by pulsed field gel electrophoresis.

Carcinoma, Squamous Cell↗

PubMed1993

Ploidy status and the response of T1 glottic carcinoma to radiotherapy.

Flow cytometric DNA ploidy measurements were performed on formalin fixed paraffin embedded tumour specimens from patients with a T1 glottic laryngeal carcinoma in order to evaluate the role of DNA content in relation to local control. From 1980 to 1987, a consecutive series of 90 patients with a T1 glottic laryngeal carcinoma were treated by radiotherapy with curative intent. Biopsies from 44 of these patients were readily available for DNA flow cytometry. In this group aneuploidy was associated with a significantly higher risk (P = 0.018) of local recurrence within 2 years after completion of radiotherapy (38% vs. 9% in the diploid group).

Aged↗

PubMed1992

Reduction of systemic exposure and toxicity of cisplatin by encapsulation in poly-lactide-co-glycolide.

The tissue distribution and normal tissue toxicity of cisplatin (cDDP) administered as poly-lactide-co-glycolide (PLAGA) microspheres, developed for loco-regional administration of cDDP to the liver, were studied in Wag/Rij rats. Venoportal administration of this formulation resulted in a reduction in total systemic and renal toxicity, which correlated with a decrease in normal tissue exposure to cDDP while maintaining high liver platinum levels. Liver-to-kidney platinum level ratios were 28 times higher after 4 h and 19 times higher after 24 h with PLAGA-cDDP microspheres than with free cDDP. Liver-to-blood platinum ratios at these times were 38 times and 36 times higher using PLAGA-cDDP. In a CC531 colon carcinoma liver micrometastases model, cytotoxicity of microsphere-released cDDP was confirmed in vivo by equal inhibition of tumor growth by PLAGA-cDDP and free cDDP over a period of 26 days. Free cDDP, however, caused significantly more histological renal damage and total body weight loss. The results were supported by the finding of higher plasma creatinine and urea concentrations 26 days after administration of free cDDP. Kidney platinum levels were 7 times lower when PLAGA-cDDP was used. These findings indicate a sparing effect on normal tissues when cDDP is targeted to the liver by formulation in PLAGA. PLAGA-cDDP microspheres may, therefore, be a useful and effective addition to current techniques of loco-regional chemotherapy for disseminated hepatic tumors.

Animals↗

PubMed1991

Fluorescent markers for hypoxic cells. A study of novel heterocyclic compounds that undergo bio-reductive binding.

The bioreductive metabolism and binding of nitroaromatic compounds has been suggested as a method for the identification of hypoxic tumour cells. Bound metabolites of suitable nitroaryl compounds (and some other reducible aromatic compounds) may fluoresce, offering an alternative to radiolabelling or NMR etc. as a diagnostic method. In this paper, the synthesis of some heteroaromatic nitro-compounds is given together with the results obtained from testing of these and other mainly nitroaromatic compounds in vitro as potential bioreductive fluorescent probes for hypoxic cells in tumours. Compounds were incubated with oxygenated or hypoxic mammalian cell suspensions for various times before evaluation of the cellular fluorescence from bioreductive metabolites by fluorescence microscopy and flow cytometry. Among those compounds yielding fluorescent metabolites in cells, considerable variation in hypoxic:oxic differential fluorescence was observed. The in vitro mammalian cell test system showed several of the compounds to be sufficiently promising to merit further investigation in vivo.

Animals↗

PubMed1991

Cell kinetic analysis of mixed populations using three-color fluorescence flow cytometry.

The development of antibodies to DNA-incorporated thymidine analogs has in turn led to the development of flow cytometric techniques for rapidly measuring cell kinetics parameters. More recently, these techniques have been applied to clinical tumor material. One problem with such measurements has been the difficulty of distinguishing malignant cells from coexistent normal cells in the biopsy material. In the present study, the feasibility of selecting out the desired malignant cell population for kinetic analysis from a mixture of cells was tested in vitro. An anticytokeratin antibody was used to discriminate between a mixture of tumor cells (cytokeratin positive) and normal cells (cytokeratin negative). The cell lines chosen for the study, A549 human lung carcinoma cells and Chinese hamster ovary (CHO) cells, were pulse labeled with iododeoxyuridine (IdUrd) and sampled every hour up to 16 hours. Selecting out cells from the mixture required the application of three-color fluorescence flow cytometry, which was carried out using the fluorochromes FITC (fluorescein isothionate, green fluorescence, IdUrd-DNA antibody), PE (phycoerythrin, orange fluorescence, cytokeratin antibody), and PI (propidium iodide, red fluorescence, DNA). This allowed single laser excitation. The staining procedure involved incubation with the IdUrd antibodies (specific antibody plus FITC-conjugated second antibody) followed by the cytokeratin antibodies (specific antibody plus PE-conjugated second antibody) and lastly by the DNA stain containing RNase. Two analysis methods of the IdUrd/DNA cytograms were applied: a mid-S window analysis and a relative movement (RM) analysis. Results of the analyses for cells selected out of mixtures were compared with results of cells stained and analyzed separately. A clear separation of the two cell lines could be obtained on the basis of orange fluorescence (cytokeratin content) despite a large overlap of their DNA histograms. By gating on high or low orange fluorescence, almost pure populations of the individual cell types could be selected out for further kinetic analysis. Little difference was seen, with both the mid-S and RM analyses, between cells gated from mixtures or stained separately. It is concluded that this technique is feasible for use on clinical material, provided good cell suspensions can be obtained, leading to the hope of increasing the accuracy of kinetic measurements on human tumors.

Animals↗

PubMed1991

Antibodies against cisplatin-modified DNA and cisplatin-modified (di)nucleotides.

Cytotoxic effects of cis-diamminedichloroplatinum-(II) (cis-DDP) are thought to be mediated by binding to DNA. Studies on binding of cis-DDP to cellular DNA rely heavily on the availability of specific antibodies. We therefore raised and characterized four rabbit antisera: one against cis-DDP-modified DNA (antiserum NKI-A59) and three others against the cis-DDP-modified (di)nucleotides cis-Pt(NH3)2d(pApG) (NKI-A68), cis-Pt(NH3)2d(GMP)2 (NKI-A10), and Pt(NH3)3dGMP (NKI-A39). Reactivities to platinum compounds were determined in an enzyme-linked immunosorbent assay (ELISA) and in a quantitative immunocytochemical assay. In the ELISA, NKI-A59 showed a high affinity for DNA heavily substituted with either cis-DDP or CBDCA [cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II)]; amounts of platinum per well giving 50% inhibition (IA50) were as low as 15 and 76 fmol, respectively. NKI-A59 also showed affinity to cis-DDP-modified poly[d(G-C)].poly[d(G-C)], poly(dC), and poly(dG). No affinity was found for trans-DDP [trans-diamminedichloro-platinum(II)]-modified DNA, enzymatically digested cis-DDP-DNA, or cis-DDP-DNA, or cis-DDP-modified poly(dA).poly(dT), oligo(dA)15.oligo(dT)15, oligo(dG)21, oligo(dG)42, or oligo(dAAAG)10. The efficiency of binding to cis-DDP-DNA decreased with decreasing DNA modification levels. Although other cis-DDP-DNA- and cis-DDP-(di)nucleotide-specific antisera have been identified, NKI-A59 is the first antiserum described that is suitable for the in situ detection of cis-DDP-DNA adducts at clinically relevant platinum levels. Adduct-specific immunostaining signals in cultured RIF-1 cells or rat liver paralleled platinum-DNA binding as measured by atomic absorption spectroscopy. The antisera NKI-A68, NKI-A10, and NKI-A39 showed high affinity for their corresponding haptens and varying affinity for non-hapten cis-DDP-DNA adducts. Their affinity for digested cis-DDP-modified DNA was up to 30 times that for intact cis-DDP-DNA. Neither NKI-A68 nor NKI-A10 resulted in specific immunocytochemical staining of cis-DDP-DNA adducts. We conclude that NKI-A68, NKI-A10, and NKI-A39 are suitable for platinum-DNA adduct analysis of digested DNA in ELISA and that NKI-A59 is suitable for platinum-DNA adduct detection at the single-cell level using immunocytochemical methods.

Animals↗

PubMed1991

Polymer-coated albumin microspheres as carriers for intravascular tumour targeting of cisplatin.

We used a poly-lactide-co-glycolide polymer (PLAGA 50:50) to formulate cisplatin (cDDP) into microspheres designed for intravascular administration. Two systems were developed. PLAGA-coated albumin microspheres and microspheres consisting of PLAGA only. PLAGA-coated microspheres displayed a mean diameter of 31.8 +/- 0.9 microns and a payload of 7.5% cDDP (w/w). Solid PLAGA microspheres exhibited a mean diameter of 19.4 +/- 0.6 microns and a payload of 20% cDDP. Release characteristics and in vitro effects on L1210 leukemia and B16 melanoma cell lines were investigated. Both types of microsphere overcame the initial rapid release of cDDP (burst effect), and PLAGA-coated albumin microspheres also showed a lag phase of approximately 30 min before cDDP release began. PLAGA-coated albumin microspheres released most of their payload through diffusion, and the coating eventually cracked after 7 days' incubation in saline supplemented with 0.1% Tween at 37 degrees C, enabling the release of any cDDP remaining. Effects of platinum, pre-released from PLAGA-coated albumin microspheres on the in vitro growth of L1210 cells were comparable with those of standard formulations (dissolved) of cDDP. Material released from non-drug-loaded PLAGA microspheres had no effect on L1210 cell growth, suggesting the absence of cytotoxic compounds in the matrix. The colony-forming ability of B16 cells was also equally inhibited by standard cDDP and pre-released drug. These studies show that formulation of cDDP in PLAGA-based microspheres prevents the rapid burst effect of cDDP seen in previous preparations and offers an improved system of administration for hepatic artery infusion or adjuvant therapy, enabling better clinical handling and the promise of a higher ratio of tumour tissue to normal tissue.

Animals↗

PubMed1991

Intratumoural administration of cisplatin in slow-release devices: II. Pharmacokinetics and intratumoural distribution.

The pharmacokinetics of cisplatin in mice with s.c. RIF 1 tumours was studied after intratumoural (i.t.) administration of drug in solution and in different slow-release devices. The data were compared with those obtained after i.p. administration of cisplatin. The slow-release devices under test were manufactured from either starch (ST) or polymeric hydrogels with different water uptakes (named T1, T2 and T3). In vitro release from these devices was approximately 100% in 2 h for starch rods, 100% in 24 h for T3 hydrogels, 45% in 4 days for T2 hydrogels and less than 10% in 4 days for T1 hydrogels. In vivo release rates agreed well with the in vitro data for T1 and T2 rods and were slightly slower in vivo for the T3 rods. The ST rods released the drug 6 times slower in vivo than in vitro. Plasma concentrations after i.t. administration were lower than those measured after i.p. administration. Systemic exposure to both total and free platinum was reduced to 70% for i.t. as compared with i.p. administration. Tumour concentrations were 4 times higher after i.t. than after i.p. administration. Tumour and peak plasma levels of platinum increased with increasing release rates. With the faster-releasing formulations (ST and T3), tumour platinum concentrations were 100 times higher than after i.p. administration. With the slower releasing formulations (T1 and T2), total tumour platinum concentrations were 2 and 9 times higher, respectively, than after i.t. administration of cisplatin in solution. Platinum distribution within the tumour was homogeneous after i.p. administration. After i.t. administration of cisplatin in solution, platinum concentrations in the centre of the tumour were approximately 4 times higher than in peripheral tumour tissue. Implantation of cisplatin in T2 and T3 hydrogel rods resulted in large concentrations of platinum in the centre of the tumour (the site of implant), which decreased steeply towards the tumour periphery. In summary, i.t. administration of cisplatin solution produced better results than did systemic (i.p.) administration in terms of tumour versus plasma drug-concentration ratios. Administration of drug in slow-release rods proved even more advantageous, although this was offset by inhomogeneous drug distributions within the tumour.

Animals↗

PubMed1991

Tumour cell repopulation during fractionated radiotherapy: correlation between flow cytometric and radiobiological data in three murine tumours.

This study tested whether the potential doubling time of tumour cells measured before or during treatment could predict the repopulation rate of surviving clonogens during fractionated radiotherapy. Tumours used for the study were a fibrosarcoma (SSK 2), an adenocarcinoma (AT 7) and a squamous cell carcinoma (AT 478), all grown subcutaneously in the C3H mouse. Potential doubling times (Tpot) were measured using the thymidine nanalogue iododexyuridine (IUdR) and flow cytometry. Results were compared with previous radiobiological studies on these tumours in which repopulation rates during radiotherapy were estimated using the tumour growth delay and tumour cure assays. Fractionated treatments consisted of daily doses of 4 or 8 Gy to clamped (hypoxic) tumours, 6 days per week for 1-3 weeks. Tpot values increased markedly during therapy for two of the tumours (SSK 2 and AT 478), by a factor of more than 10 for AT 478 in the third treatment week. Tpot remained approximately constant for the third tumour (AT 7). In no case was there evidence from the labelling studies of a shortening of Tpot which would suggest accelerated repopulation. From the radiobiological data, effective clonogen doubling times during radiotherapy were calculated from the doses required to produce a given effect in short and long treatment schedules. In the second week of treatment, effective clonogen doubling times in two tumours were approximately equal to the pretreatment Tpot, and shorter than the pretreatment Tpot in the third tumour. At some time during treatment, the surviving clonogens in these tumours therefore proliferated at the same rate or faster than before treatment. The difference between the labelling and radiobiological measurements was ascribed to the fact that, shortly after the start of a fractionated treatment, the IUdR labelling technique measures primarily doomed cells. These results show that kinetic measurements using DNA labelling techniques made during fractionated radiotherapy in most cases do not reflect the proliferation status of the surviving cells which are responsible for treatment outcome. Pretreatment Tpot measurements give a much better indication of the proliferation rate of surviving cells but in some cases may underestimate repopulation during radiotherapy.

Adenocarcinoma↗

PubMed1991

Formation of interaction products of carboplatin with DNA in vitro and in cancer patients.

Binding of the cytostatic drug carboplatin to DNA was studied in solution, in RIF-1 and CHO cell lines and in human buccal cells after in vitro or in situ drug exposure. Results were compared with DNA adduction by cisplatin. The rate of binding in solution, determined by atomic absorption spectroscopy, was 35 times lower for carboplatin than for cisplatin. Adduct formation in cells in vitro was determined in a quantitative immunostaining assay. Staining intensities after carboplatin treatment were at least 29 times lower than after an equimolar dose of cisplatin. For RIF-1 and CHO cells, maximum levels of carboplatin-induced DNA modification were obtained 24 h after treatment; these levels correlated with cell killing. Adduct-specific staining in buccal cells from two carboplatin-treated patients increased 5-7 fold between 0 and 14 h after infusion, reaching a maximum at 10-14 h. This strongly contrasts with buccal cells from a cisplatin-treated patient, in which the adduct-specific staining signal increased by only 23% between 0 and 6 h after infusion, and then declined. This difference in the rate of adduct formation in vivo is consistent with the in vitro data.

Animals↗

PubMed1990

Correlation between cell killing by cis-diamminedichloroplatinum(II) in six mammalian cell lines and binding of a cis-diamminedichloroplatinum(II)-DNA antiserum.

The relationship between cell killing and the binding of the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP) to DNA was studied in six mammalian cell lines. Two of the human cell lines (COV413B) were of the same origin, comprising one sensitive to cis-DDP and the other with induced resistance to the drug. The four other lines, two rodent (RIF-1, Chinese hamster ovary) and two human (A2780, A1847), were unrelated. The cell lines differed in their sensitivity to cis-DDP, as tested in a clonogenic assay. cis-DDP-DNA binding was determined by quantitative immunocytochemistry using an antiserum against cis-DDP-modified DNA. The resistance factors relative to RIF-1, calculated from full survival curves for cis-DDP, were 3.8 +/- 0.4 for Chinese hamster ovary cells and 8.8 +/- 0.7 for both A2780 and A1847 lines. Using quantitative immunocytochemistry, the levels of the adduct-specific nuclear staining density compared with RIF-1 cells were 4.8 +/- 0.2 for Chinese hamster ovary cells, 9.1 +/- 0.2 for A2780, and 10.0 +/- 0.1 for A1847 cells, i.e., in good agreement with the resistance factors. In studies with the COV413B cells and their cis-DDP-resistant counterpart COV413B-PtR, immunologically detected adduct levels again correlated closely with resistance factors (correlation coefficient = 0.97). The kinetics of cis-DDP-DNA adduct formation and loss was investigated in RIF-1, A2780, and A1847 cells by the immunocytochemistry technique. Adduct levels after a 1-h incubation with approximately equitoxic doses of cis-DDP increased by 18 to 32% (average, 27%) between 0 and 6.5 h after treatment and then declined. Adduct half-lives in this latter phase did not correlate with the sensitivities of the cells for cis-DDP. These results indicate that the initial level of cis-DDP-DNA binding measured by quantitative immunocytochemistry may be a reasonable predictor of sensitivity to this chemotherapeutic drug.

Animals↗

PubMed1990

A new intraperitoneal tumor model in the rat.

A new tumour model that is particularly suitable for testing intraperitoneal chemotherapy is described. Single tumours were induced to grow in the mesentery of rats by the implantation of small pieces taken from subcutaneous tumours. Tumour growth was monitored by repeated laparotomies at which the tumour size was measured with calipers. In this way, growth curves of treated and untreated tumours could be defined. The diameter of untreated intraperitoneal tumours increased linearly with time [diameter (mm) = 0.39 t (days) +2.4]. Tests using different numbers of laparotomies showed that the procedure itself had little influence on growth. Cell kinetic studies of 6-mm tumours showed a mean labelling index of 31% and a volume-doubling time of 3.9 days, resulting in cell-loss factors probably in excess of 70%. The model was tested by assessing the effect of the chemotherapeutic agent cisplatin. Regression and regrowth could be satisfactorily followed, leading to estimates of growth delay. This model therefore provides a quantitative way to assess the response of intraperitoneal tumours to chemotherapy.

Adenocarcinoma↗

PubMed1990

Intratumoural administration of cisplatin in slow-release devices. I. Tumour response and toxicity.

In this study we investigated the effect of the incorporation of cisplatin in slow-release systems on tumour response and animal toxicity after intratumoural (i.t.) administration. Solid slow-release rods with incorporated cisplatin were prepared either from starch or from three different polyether-hydrogel formulations. In vitro release rates from these rods were widely different. With the starch system, approximately 100% release was obtained in 2 h. For the hydrogel formulations, release was approximately 100% in 1 day for a formulation with 40% water uptake (T3), 45% within 4 days for a formulation with 14% water uptake (T2) and 8% within 4 days for a formulation with 4% water uptake (T1). The slow-release rods containing graded amounts of cisplatin were implanted i.t. in s.c. RIF1 murine tumours. The i.t. administration of cisplatin in starch rods did not reduce animal toxicity or increase tumour response relative to i.t. injections of cisplatin in solution. For the hydrogel rods, the tumour response and animal toxicity for a given dose of cisplatin decreased with decreasing release rate. Higher doses of cisplatin could therefore be delivered with the slower-releasing hydrogel formulations. The slowest-release hydrogel rods (T1) had very little effect on either tumour (growth delay) or host (animal weight loss), even at cisplatin doses 8 times that tolerated as an i.p. injection. The fast (T3)- and intermediate (T2)-release hydrogel rods resulted in dose dependent tumour growth delays that were longer than those obtained with i.p. or i.t. administration of cisplatin. The highest response, a tumour growth delay of 55 days, was obtained with the intermediate-release hydrogel rods (T2) at a cisplatin dose of 40 mg/kg. Analysis of tumour growth delay for a given level of toxicity indicated that the intermediate-release formulation (T2) was slightly better than the fast-release formulation (T3) and confirmed the therapeutic advantage of i.t. implants over systemic therapy.

Animals↗