Combination of sunitinib, cetuximab and irradiation in an orthotopic head and neck cancer model.

BACKGROUND: Recent preclinical and clinical studies indicate beneficial effects from combining radiotherapy with either anti-angiogenic drugs or anti-epidermal growth factor receptor (EGFR)-targeting agent. To investigate the effect of combining these approaches, we evaluated in vivo the antitumor efficacy of the anti-angiogenic compound sunitinib, an oral, multi-targeted tyrosine kinase inhibitor that inhibits among others vascular endothelial growth factor (VEGF) receptors-1, -2 and -3, cetuximab, a mAb targeting the EGFR, and irradiation (RT) given alone and in combination. MATERIALS AND METHODS: Investigations were carried out using a VEGF-secreting human head and neck tumor cell line, CAL33, with a high EGFR content, growing as orthotopic xenografts in nude mice. Three days after tumor cell injection, sunitinib (20 mg/kg, p.o.), cetuximab (1 mg/kg, i.p.), both 5 days/week seven doses, and RT (6 Gy, 3 days/week, four doses) were administered alone and in combination during 9 days. RESULTS: Concomitant administration of drugs produced a marked and significant supra-additive decrease, and the addition of RT completely abolished tumor growth. The drug association markedly reduced tumor cell proliferation (Ki67) and the number of the vessels, but enhanced cell differentiation. CONCLUSION: The efficacy of this combination of sunitinib, cetuximab and RT may be of clinical importance in the management of head and neck cancer patients.

Combined effects of bevacizumab with erlotinib and irradiation: a preclinical study on a head and neck cancer orthotopic model.

Clinical benefit has been demonstrated in patients with head and neck tumours receiving an anti-epidermal growth factor receptor (EGFR) agent in combination with radiotherapy (RT). Recent preclinical and clinical studies suggest beneficial effects from combining anti-angiogenic drugs with RT. To investigate the effect of combining these approaches, we evaluated in vivo the anti-tumour efficacy of the anti-angiogenic compound bevacizumab, a highly specific monoclonal antibody directed against the vascular endothelial growth factor (VEGF), erlotinib, an EGFR tyrosine kinase inhibitor, and irradiation given alone and in combination. Investigations were performed using a VEGF-secreting human head and neck tumour cell line, CAL33, with a high EGFR content, injected as orthotopic xenografts into the mouth floor of nude mice. Three days after tumour cell injection, bevacizumab (5 mg kg(-1), 5 days a week, i.p.), erlotinib (100 mg kg(-1), 5 days a week, orally) and irradiation (6 Gy, 3 days a week) were administered alone and in combination for 10 days. As compared with the control, concomitant administration of drugs produced a marked and significant supra-additive decrease in tumour mass; the addition of irradiation almost completely abolished tumour growth. The drug association markedly reduced the number of metastatic nodes and the triple combination significantly reduced the total number of pathologically positive lymph nodes as compared with controls. The RT-induced proliferation, reflected by Ki67 labelling, was reduced to control level with the triple combination. Radiotherapy induced a strong and very significant increase in tumour angiogenesis, which was no longer observed when combined with erlotinib and bevacizumab. The efficacy of the combination of bevacizumab+erlotinib and RT may be of clinical importance in the management of head and neck cancer patients.

Activation of nuclear factor kappaB through the IKK complex by the topoisomerase poisons SN38 and doxorubicin: a brake to apoptosis in HeLa human carcinoma cells.

The transcription factor nuclear factor (NF) kappaB is involved in the regulation of cell survival. NFkappaB is activated in many malignant tumors and seems to play a role in the resistance to cytostatic treatments and escape from apoptosis. We have studied the effects on NFkappaB activation of two topoisomerase poisons and DNA damaging agents that are used in chemotherapy: SN38 (7-ethyl-10-hydroxycamptothecin), the active metabolite of CPT11, and doxorubicin. In HeLa cells, both drugs activate NFkappaB using a preexisting pathway that requires a functional IkappaB-specific kinase complex, IkappaB-specific kinase activation, IkappaB-alpha phosphorylation, and degradation. Blocking NFkappaB activation by stable expression of a mutant super-repressor IkappaB-alpha molecule sensitized HeLa cells to the apoptotic actions of drugs and tumor necrosis factor. RNase protection assay analysis demonstrate that NFkappaB is involved in the regulation of a complex pattern of gene activation and repression during the cellular response of HeLa cells to topoisomerase poisons. The blockade of NF-kappaB activation seems to shift the death/survival balance toward apoptosis.

Establishment, characterization, chemosensitivity, and radiosensitivity of two different cell lines derived from a human breast cancer biopsy.

In vitro culture of a human breast cancer biopsy fragment gave rise to two permanent cell lines, CAL 18 A and CAL 18 B, which were differentiated by both morphological and ultrastructural analysis. The karyotypic and growth properties of these two cell lines also differed, providing further evidence of cell heterogeneity within a given tumor. Both cell lines lost their hormone receptors in vitro. CAL 18 A cells grew in agar and were tumorigenic after inoculation into nude mice; neither of these properties was observed in CAL 18 B cells. The chemosensitivity of 12 antineoplastic drugs was assessed by a short-term assay, using inhibition of tritiated thymidine incorporation by the cells after contact with the drugs as the end point. Only a few drugs were active at moderate concentrations. The overall responses of both cell lines were similar. The cell survival curves, established by the colony method following a single dose of radiation, were also very similar, despite the greater heterogeneity of CAL 18 B cells. The two cell lines appear to be interrelated, since CAL 18 B cells were occasionally observed to emerge from CAL 18 A clones, suggesting that malignant cell redifferentiation may occur spontaneously in vitro.

Response to fluorouracil therapy in cancer patients: the role of tumoral dihydropyrimidine dehydrogenase activity.

PURPOSE: The aim of the present study was to analyze the role of thymidylate synthase (TS; main cellular target of fluorouracil [FU]) and dihydropyrimidine dehydrogenase (DPD; rate-limiting enzyme of FU catabolism) in tumoral biopsies with respect to FU responsiveness. PATIENTS AND METHODS: This prospective study was conducted on 62 head and neck cancer patients (six stage II, 16 stage III, and 40 stage IV). All received first-line chemotherapy with biomodulated FU (5-day continuous infusion). Before treatment, a tumor biopsy and control biopsy (symmetrical nontumoral area) were obtained. Cytosolic TS and DPD activities were measured using radioenzymatic assays. RESULTS: DPD activity was detectable in all samples, without a significant difference between tumoral (median, 60 pmol/min/mg protein; range, 13 to 193) and nontumoral samples (median, 68 pmol/min/mg protein; range, 12 to 150). Tumoral TS and tumoral DPD were not significantly influenced by tumor localization or tumor staging. Among 52 tumors assessable for clinical response, we observed 46% complete responses (CRs), 33% partial responses (PRs), and 21% no responses (NRs). No relationship was demonstrated between TS activity and response to FU therapy. The comparison of tumoral DPD between complete responders and partial or nonresponders showed a trend toward significance (P = .06). In an attempt to reduce variability, we analyzed the tumoral/nontumoral DPD activity ratio; complete responders exhibited a significantly lower normalized DPD than partial or nonresponding patients (median, 0.86, 1.18, and 1.42 for CR, PR, and NR, respectively; CR v PR plus NR, P = .03). CONCLUSION: Although resistance to FU is multifactorial, the present clinical study suggests that FU catabolism in target cells is probably a determinant factor for FU responsiveness in cancer patients and justifies the clinical use of specific DPD inhibitors as FU biomodulators.

Search for the optimal schedule for the oxaliplatin/5-fluorouracil association modulated or not by folinic acid: preclinical data.

The combination of oxaliplatin (LOHP)-5-fluorouracil (FU)-folinic acid (FA) has provided high response rates in pretreated patients with advanced colorectal cancer that is resistant to FU-FA. However, the choice of the optimal schedule between LOHP, FU, and FA remains open. The purpose of the present study was to compare, at equivalent drug area under the curve, different schedules for the LOHP-FU +/- FA combinations on four human colorectal cancer cell lines. FU +/- FA was tested as a 2-h short exposure ("bolus"), a 118-h continuous exposure ("infusion"), or a 22-h mixed exposure ("De Gramont protocol"). LOHP was administered for 2 h before, during, or after FU +/- FA exposure. Isobologram analyses revealed that LOHP associated with FU +/- FA resulted in synergistic cytotoxic effects whatever the tested schedules (in > or = 75% of cases). For the FU-LOHP combination, cytotoxicity was significantly different according to the FU exposure type (short > mixed > continuous) and was independent of the LOHP position. In contrast, for the FU-FA-LOHP combination, neither the FU exposure type nor the LOHP position significantly influenced cytotoxicity. The presence of FA significantly enhanced the cytotoxicity of FU-LOHP (P < 0.001); this potentiation was independent of the FU exposure type and was significantly influenced by the LOHP position (LOHP after FU-FA > LOHP during FU-FA > LOHP before FU-FA). In conclusion, in contrast with the recognized superiority of continuous FU exposure over short exposure when the drug is given alone, the FU-LOHP combination is more cytotoxic when FU is given as a short exposure. This suggests the potential interest of such a schedule in the clinical setting.

Dihydropyrimidine dehydrogenase: a tumoral target for fluorouracil modulation.

Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme of 5-fluorouracil (FU) catabolism. Ethynyluracil (776C) is a very potent, mechanism-based irreversible DPD inhibitor that improves the antitumor efficacy and the therapeutic index of FU in laboratory animals. We tested the cytotoxic effects of the FU-776C combination on a panel of 12 human cancer cell lines (4 breast, 4 head and neck, 3 colon, and 1 duodenum). Basal DPD activity (radioenzymatic assay) and FU sensitivity [FU 50% inhibitory concentration (IC50), 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test] were determined. The FU potentiation by 776C was calculated from the ratio (F) of FU IC50 without 776C divided by FU IC50 with 776C. 776C was not cytotoxic to any of the cell lines tested. On CAL51 cell line expressing a high basal DPD activity, FU enhancement by 776C was a saturable phenomenon related to the 776C concentration; the inhibition of DPD increased between 10(-12) to 10(-6) M of 776C. For the following studies, 776C was tested at 10(-6) M. FU IC50 varied from 15 to 7770 microM among cell lines (median, 390 microM). Basal DPD activity ranged from not detectable (< pmol/min/mg protein) to 320 pmol/min/mg protein among cell lines (median, 53 pmol/min/mg protein). For the 12 cell lines tested, the mean F ranged from 0.7 (no enhancement of FU cytotoxicity by 776C) up to 5.2 and was significantly related to the basal DPD activity: the greater the DPD activity, the greater the FU enhancement factor (Spearman rank correlation, P = 0.019). Enhancement of FU cytotoxicity by 776C occurred only in the six cell lines expressing the greatest basal DPD activity (>50 pmol/min/mg protein, F ranging between 1.7 and 5. 2), whereas 776C did not modify FU cytotoxicity in the remaining cell lines expressing the lowest DPD activity (<50 pmol/min/mg protein, F ranging between 0.7 and 1.4). F was significantly different between these two groups of cell lines (P = 0.005). These results point out that DPD is an interesting target for FU pharmacomodulation.

Effects of prolonged exposure to cisplatin on cytotoxicity and intracellular drug concentration.

The present study was designed to analyse the cytotoxic effect of cisplatin in vitro as a function of various exposure times (up to 120 h), keeping constant the parameter C x T (product of the drug concentration per time). Intracellular drug concentrations were measured in parallel following analysis of cisplatin influx and efflux characteristics. A head and neck cancer cell line was selected to represent the spectrum of cisplatin antitumour activity. The IC50 values (micrograms/ml) for 1, 2, 11 and 121 h were, respectively 4.51, 2.73, 0.27 and 0.151. Reduction of the IC50 was clearly not linearly related to prolongation of the cisplatin exposure time. The kinetics of cisplatin incorporation into CAL 27 cells was investigated as a function of different cisplatin concentrations. A plateau was reached after 16 h of contact. For the extracellular cisplatin concentrations of 1, 2.5, 5 and 10 micrograms/ml, the average intracellular Pt concentrations at the plateau were, respectively (ng/10(6) cells): [mean (S.D.)] 12.8 (0.98), 31.11 (5.12), 71.38 (6.03) and 136.7 (16.5). Intracellular Pt concentrations were linearly related to the extracellular drug concentration (r = 0.99). The drug left the cells following a two-slope kinetics pattern with an alpha half-life of 1.29 h and a beta half-life of 94.4 h. The cytotoxic effect for a given C x T clearly differed for the different cisplatin exposure times. The longest exposure time (121 h) gave the least pronounced cytotoxicity. The intracellular Pt concentrations were linearly related to the C x T values. Cisplatin levels were much lower after the 121 h exposure. These data may prove valuable in establishing a rationale which can aid in selection of optimal modes of clinical cisplatin administration.

Epidermal growth factor receptor expression and suramin cytotoxicity in vitro.

Twenty-five cell lines derived from nine different human cancers were tested for the cytotoxic activity of suramin. Two different initial cellular concentrations were used: C1 (800-2000 cells per well) and C2 (3000-7000 cells per well). Suramin concentrations ranged from 50 to 2500 micrograms/ml. Cytotoxicity was assessed by the MTT test. Epidermal growth factor receptors (EGFR) were assayed by competition analysis and Scatchard plots. In sixteen cell lines suramin had an unexpected growth stimulation effect at low concentration (50-125 micrograms/ml). IC50 varied from 21 micrograms/ml (osteosarcoma, OS2) to 1408 micrograms/ml (melanoma, CAL 24) and, within melanoma cell lines, it varied from 120 micrograms/ml (CAL 41) to 1408 micrograms/ml (CAL 24). The individual IC50 values were positively and significantly linked with the initial cellular density. Eighteen cell lines had measurable EGFR (six with two families of sites, twelve with one): Kd varied between 0.004 nmol/l for the highest affinity site (melanoma, CAL 7) to 1.852 nmol/l for the lowest affinity site (lung, CAL 12). There was no relation between presence or absence of EGF binding sites and distribution of IC50, but for cells with measurable EGFR there was a weak but significant correlation between the number of EGF binding sites per cell and the corresponding IC50 (r = -0.53, P = 0.021).

Importance of the irradiation timing within a chemoradiotherapy sequence including cisplatin and 5-FU-folinic acid. Experimental results.

The objective of the present in vitro study was to determine an optimal timing of the irradiation in the combination cisplatin (CDDP) and 5-fluorouracil-folinic-acid (5-FU-FA) allowing a maximal cytotoxic effect on a human cell line derived from a head and neck carcinoma (CAL 27 cells). The various tested chemoradiotherapy sequences were applied in parallel to human keratinocytes in culture (SVK 14 cells). This was done in order to define the best sequence allowing the achievement of an optimal selectivity of the cytotoxic effects. The drug sequence was: CDDP over 2 h then fresh medium was added including the tandem 5-FU-d,I FA applied 6 h after CDDP, for 5 days. Irradiation was applied only once and at various times within the drug sequence. The cytotoxicity effects of the different chemoradiotherapy combinations were assessed by the MTT semi-automated test. The part taken by the 5-FU-FA combinations in the overall cytotoxicity was examined; an effect was apparent on CAL 27 cells only. The evolution of the radiation effect (RE = cell survival after drugs/cell survival after drugs plus irradiation) was analysed as a function of the different times of irradiation within the given drug sequence. Clearly, the RE values were dependent upon time at which the radiation dose in the chemoradiotherapy regimen was administered. For CAL 27 cells, irradiation effects were maximal at the first irradiation time tested after the end of the CDDP exposure (i.e. t = 3.5 h). In contrast, this optimal chemoradiotherapy timing for better cytotoxicity on CAL 27 cells did not correspond to that of SVK 14 cells. Consequently, it was possible to establish that the best time for the selectivity index was located shortly after the CDDP exposure.

Relevance of tumoral folylpolyglutamate synthetase and reduced folates for optimal 5-fluorouracil efficacy: experimental data.

The purpose of this study was to investigate folate-related predictors of 5-fluorouracil (5-FU) cytotoxicity in the presence or absence of l-folinic acid (l-FA). Intracellular concentrations of the reduced folates (tetrahydrofolate + 5,10-methylenetetrahydrofolate) and folylpolyglutamate synthetase (FPGS) activity were determined in 14 human cancer cell lines expressing a spontaneous sensitivity to 5-FU. On these 14 cell lines grown without l-FA supplementation, a significant positive correlation was demonstrated between basal intracellular folate concentration and FPGS activity. 5-FU sensitivity (IC50 range 0.6-25.4 microM) was not related to the basal intracellular folate concentration, whereas, significantly, it was linked to FPGS activity (range 2.5-11.1 pmol/min/mg protein): the higher the FPGS activity, the greater the 5-FU sensitivity. Under l-FA supplementation (0.01-300 microM), intracellular reduced folates increased continuously without evidence of saturation in all cell lines; the pattern of accumulation was independent of the FPGS activity. l-FA enhanced 5-FU cytotoxicity by a factor of 1.9-6.4 in 12 of the 14 cell lines. In the 12 FA-sensitive cell lines, the l-FA concentrations allowing 90% of maximum 5-FU potentiation [l-FA]90 ranged between 0.7 and 107.9 micro M (median 1.9); in contrast, the intracellular concentrations of reduced folates allowing 90% of maximum 5-FU potentiation were much less variable (range 7.6-38.3, median 24.8 pmol/mg protein). In the presence of [l-FA]90, 5-FU sensitivity remained significantly correlated to the basal FPGS activity. In addition, reduced folates were measured in 96 tumoral samples (50 head and neck, 16 colon, 30 liver metastases from colorectal cancer) taken before treatment. Almost all investigated tumours had folate concentrations below the median concentration required for optimal 5-FU potentiation in vitro: median levels (range, pmol/mg protein) were 3.8 (0-17.7) for head and neck, 5.8 (2.3-12.0) for colon and 12.1 (1.7-118.5) for liver metastases. Above all, these data establish the relevance of FPGS activity for predicting the efficacy of 5-FU modulated by FA or not and point to the potential clinical interest of FPGS determination in human tumours.

A role for dihydropyrimidine dehydrogenase and thymidylate synthase in tumour sensitivity to fluorouracil.

Despite being one of the oldest anti-cancer drugs, fluorouracil (FU) is still being increasingly used in cancer chemotherapy. The source of variability for FU sensitivity in patients may be complex, although an overproduction of thymidylate synthase (TS) was the only mechanism of resistance identified in tumours from FU-resistant patients. Dihydropyrimidine dehydrogenase (DPD) is the first and rate-limiting enzyme of FU catabolism. Thus, DPD activity may be a potential factor for controlling FU responsiveness. A panel of 19 human tumour cell lines, including digestive tract, breast and head and neck cancer cells, were investigated. Both TS and DPD activities were measured in parallel to FU responsiveness. None of the cell lines had been previously exposed to FU, and thus expressed a spontaneous sensitivity to FU. Sensitivity between cell lines showed marked differences, with IC50 values ranging from 45 ng/ml (colon cell line) to 5063 ng/ml (head and neck cell line). TS activity was measurable in all cell lines and varied within a 46-fold range. DPD activity was detected in all but four cell lines, showing a 100-fold range of variation. Cell lines most sensitive to FU exhibited the lowest DPD and TS activities and vice versa. Simple linear regression analysis showed that both TS (r2 = 0.22, P = 0.042) and DPD (r2 = 0.27, P = 0.022) activities were significantly correlated to FU effectiveness (log 10 IC50): the greater the enzyme activities, the higher the FU IC50. TS and DPD were demonstrated to be independent variables. A multiple regression analysis showed that the combination of TS and DPD activities explained 36% of the variability in FU IC50 (r2 = 0.36, P = 0.01). Two groups of cell lines could be identified, one group with both low TS and low DPD activities (G1), and the other with either high TS and/or high DPD activities (G2). Mean FU IC50 values were 193 and 930 ng/ml in G1 and G2, respectively, and this difference in FU sensitivity was highly significant (P = 0.009). The present study shows, for the first time, that DPD activity in tumour cells is an independent factor significantly related to FU sensitivity. These results should encourage DPD and TS coupled measurements in tumours of patients before FU treatment in order to establish their prognostic relevance. DPD and TS measurements could also be used during the treatment course to determine the implication of these enzymes in the development of tumour resistance to FU.

Wide range for optimal concentration of folinic acid in fluorouracil modulation--experimental data on human tumour cell lines.

The clinical use of the fluorouracil (FU)-folinic acid (FA) combination is hampered by the still open choice of the optimal schedule, with marked controversy as concerns the optimal FA dose. This in vitro study on FU-FA combinations in 17 human cancer cell lines, representative of tumour types responding to FU-FA treatment, reassesses the notion of the optimal FA concentration. Cells were exposed for 5 days to various FU-FA concentrations (0.07-77 microM, 14 concentrations, for FU; and 0.0025-100 microM for FA). The growth inhibition was assessed by the MTT test. The investigated cell lines exhibited FU IC50 ranging from 0.4 to 38.9 microM (median 3.7 microM). In six out of 17 cell lines investigated, the addition of FA did not result in a substantial enhancement of FU cytotoxicity (group 1). For the remaining 11 cell lines responding to FA supplementation (group 2), the maximal enhancement factor ranged from 3 to 8, meaning that in the presence of optimal FA concentration, the efficient FU concentration (IC50) was reduced by between 3 and 8 as compared to the efficient FU concentration without FA supplementation. For cell lines responding to FA supplementation, the optimal FA concentrations ranged from 10(-7) to 4 x 10(-4) M (4000-fold range) with a median value at 9.6 x 10(-7) M. Distribution of cell doubling time was not significantly different between group 1 and group 2. In contrast, the FU IC50 were significantly different (P = 0.02) between group 1 (median 7.4 microM) and group 2 (median 2.2 microM), thus indicating that cell lines with the greatest FU cytotoxicity enhancement by FA were those intrinsically sensitive to FU and vice versa.

Tamoxifen enhances the cytotoxic effects of the nitrosourea fotemustine. Results on human melanoma cell lines.

Fotemustine (Fote) is a new amino acid-linked chloroethyl nitrosourea which has been shown to be useful in disseminated malignant melanoma. The aim of the present study was to analyse the cytotoxic effects resulting from the combination of anti-oestrogens and Fote on human melanoma cell lines. The anti-oestrogens tested were tamoxifen (TMX, 5 x 10(-7) mol/l and 5 x 10(-6) mol/l) and 4OH TMX (5 x 10(-8) mol/l and 5 x 10(-7) mol/l). As a preliminary step, a series of nine human melanoma cell lines was screened in order to identify and quantify the presence of oestradiol receptors (ER) in these cell lines. This led to the selection of an ER-positive (+) cell line. The drugs alone or in combination were then tested against CAL 1 ER (+) and CAL 7 ER (-) melanoma cell lines. Different sequences of drug combinations were tested using clinically compatible drug concentrations. For CAL 1 cells, there was a growth inhibitory effect induced by the anti-oestrogens given alone. Overall, the presence of the anti-oestrogens resulted in higher cytotoxic effects than when cells were exposed to Fote alone. The lowest IC50 Fote values as compared to Fote alone were generated by the sequences in which the anti-oestrogens were administered before Fote. Significantly, these associations with anti-oestrogens enabled the IC50 values of Fote to be reduced by up to 80%. Globally, TMX and 4OH TMX had similar synergistic effects. TMX and 4OH TMX had a modest influence on Fote cytotoxic effects against CAL 7 ER-negative cells. These data may be useful for optimal planning of future clinical trials for malignant melanoma using anti-oestrogens and nitrosoureas.

Evaluation of the time-schedule dependency for the cytotoxic activity of the new vinca alkaloid derivative, S 12363 (vinfosiltine).

S 12363 is a new vinca alkaloid derivative obtained by appending an optically active alpha-aminophosphonate at the C23 position of 04-deacetyl vinblastine. The present study concerns four different human tumour cell lines, which represent the spectrum of vinca alkaloid clinical activity. The influence of time exposure on S 12363 growth inhibition was studied in vitro. Cells were exposed to the drug during the following exposure times: 5, 15, 30 min and 1, 3, 6, 12, 24, 48, 72, 144 h. The concentrations of S 12363 applied were between 1 x 10(-2) and 1 x 10(3) nmol/l. The cytotoxic effects were assessed by using the methyltetrazolium (MTT) semi-automated test. Considering the IC50 values in terms of concentration (C) x time (T), I (C x T)50, it was shown that for an equal growth inhibitory effect (50% of cell death) the increased exposure times required higher cumulative drug exposures. More precisely, only very long exposure (greater than 24 h) resulted in very high I (C x T)50. The drug exposure ratios which correspond to I (C x T)50 values for 144 h divided by the I (C x T)50 values for 0.25 h ranged between 2.8 and 18.3. If T and C had symmetrical effects on the final growth inhibition, the I (C x T)50 ratios should have been equal to one. For all cell lines investigated there were similar dose-response curves following two types of S12363 exposure: a single day exposure or three successive daily exposures, the total C x T values being the same in both experimental situations. The basic pharmacological information provided by the present study may encourage further clinical trials of this potentially interesting new vinca alkaloid.

Steroid receptors in human osteoblast-like cells.

The presence and functions of steroid receptors were evaluated in three human osteosarcoma cell lines (OS1 = SA OS; OS2 = HOS TE 85, and OS3 = MNNG HOS TE 85). The human breast cancer cell line MCF-7 was used as internal control for oestrogen receptors (E2R). High and low affinity sites were characterised. The high affinity sites had a similar dissociation constant in all four cell lines. In contrast, the number of sites per cell was higher in MCF-7 cells. E2 did not significantly modify the number of progesterone receptors (PgR) per cell in any of the osteosarcoma lines. As expected, E2 increased the number of PgR sites per MCF-7 cell. 4-hydroxytamoxifen decreased the growth of MCF-7 cells only. OS1 and OS2 were sensitive only to the highest concentration tested, which produces only non-specific cytotoxic effects. Thus E2R and PgR were found in osteoblast-like cells, but the function of E2R in such cells remains unknown.

Doxorubicin weekly low dose administration: in vitro cytotoxicity generated by the typical pharmacokinetic profile.

The cytotoxic effects of prolonged exposure to low concentrations of doxorubicin or a doxorubicin bolus were examined in vitro on four human breast cancer cell lines to simulate the plasma concentration profile of weekly low-dose (WLD) doxorubicin in breast cancer patients. Cells were exposed to doxorubicin for various prolonged times (24, 72, 120 and 192 h) and with different drug concentrations (5, 10, 20, 50 and 80 nmol/l). In a series of parallel experiments, cell lines were placed in contact with the drug for short periods (1 h) before prolonged exposure to doxorubicin; the concentrations of these pulses were 150, 250 and 350 nmol/l. A constant decrease in tritiated thymidine incorporation was noted as a function of the drug concentration and the duration of the cell contact with the drug. Interestingly the lowest concentrations (5-10 nmol/l) produced marked cytotoxic effects. For equivalent concentration x time values, experiments including doxorubicin pulses resulted in greater cytotoxicity than continuous exposure alone, in a dose-related manner. This finding was related to differences in intracellular doxorubicin concentrations. Results suggest that the rather empirically designed WLD doxorubicin schedule can generate greater cytotoxic effects than continuous doxorubicin administration alone.

Cytotoxic effects of long-term circulating ultrafiltrable platinum species and limited efficacy of haemodialysis in clearing them.

We applied haemodialysis to clear platinum (Pt) circulating species following renal insufficiency due to an accidental cisplatin overdosage (205 mg/m2 instead of 100 mg/m2). Serum samples were repeatedly obtained during this clinical episode from day 5 up to day 30 after cisplatin dosing. A serum aliquot taken at day 22 after cisplatin administration was tested to assess the possible cytotoxicity exhibited by the circulating Pt species on a head and neck tumour cell line. The profile of ultrafiltrable (UF) Pt during successive haemodialysis cycles was striking. After each haemodialysis cycle, a marked decrease in UF Pt, occurred but was followed by more or less pronounced rebounds. Cisplatin concentration-cytotoxic effect curves obtained in vitro from patient serum before cisplatin administration and healthy control serum exhibited very similar concentration effect profiles. In contrast, the patient serum taken at day 22 after cisplatin administration resulted in marked cytotoxic effects, which were much greater than those which could have been anticipated considering the Pt concentration of this serum sample. The present report underlines the limited usefulness of haemodialysis for rescuing cisplatin treated patients, exhibiting unanticipated postinfusion renal failure with overexposure to the drug. The in vitro investigations suggest that pharmacological effects of Pt derivatives may not only be attributable to short-term effects of the drug diffusion into tissues, but also to more delayed effects from Pt circulating species.

Sequence studies on the 5S RNA of Proteus vulgaris: comparison with 5S RNA of Escherichia coli.

We have studied the sequence of 5S ribosomal RNA of Proteus vulgaris. Although several doubts remain, it seems that no more than 8 per cent of the nucleotides differ from those of Escherichia coli 5S RNA. Of the ten bases differing from Escherichia coli 5S RNA sequence, three changes take place in one of the six positions which are known as intercistronic mutation points in Escherichia coli 5S RNA. We can conclude that they are "hot spots" of mutation.

Comparative study of the 16S RNA's of Escherichia coli and Proteus vulgaris.

We have studied the primary structure of 16S ribosomal RNA from Proteus vulgaris. The oligonucleotides containing methylated bases appeared to be the same as those of Escherichia coli, with one exception. We have also studied the base composition of the oligonucleotides obtained after T1 ribonuclease digestion of 16S RNA. On the basis both of their position on the fingerprint and of their pancreatic ribonuclease analyses, approximately 25 appeared to differ from those found in the E. coli T1 fingerprints. From the isolation of large fragments arising from the action of endogeneous endonucleases, we have concluded that the RNA sequences of both species are very similar. We have shown that the 5' and 3' extremities of 16S RNA are mostly conserved. It appears that the regions which are known to interact with ribosomal proteins in E. coli (particularly S8 and S15) are also less modified. It is noteworthy that the sequence modifications which have been observed are clustered and often correspond to regions of heterogeneity in E. coli 16S RNA.