Stephen B. Howell

cis-Diamminedichloroplatinum(II) Accumulation in Sensitive and Resistant Human Ovarian Carcinoma Cells

SummaryWe examined the short-term accumulation of cisplatin (DDP) in sensitive 2008 human ovarian carcinoma cells and in a 2- to 3-fold DDP-resistant and accumulation-deficient variant. During the 1st min of exposure to 500 μM DDP, sensitive cells accumulated platinum at a rate of 187±63 pmol/mg protein per min, whereas resistant cells accumulated platinum at 123±85 pmol/mg protein per min, a rate that was 66% that of sensitive cells. From 2–10 min of exposure, sensitive and resistant cells accumulated the drug at rates of 51.4±21.5 and 34.0±9.70 pmol/mg protein per min, respectively. In resistant cells, this rate again represented 66% that of sensitive cells. For each cell line, the DDP accumulation was 3.6 times faster during the 1st min that it was over 2–10 min. Initial DDP accumulation was linear with drug concentration in each cell line. Efflux measurements were made over a 50-min period after a 10-min load in 500 μM DDP. The loss of platinum was biphasic in each cell line, with an initial, rapidly effluxing component being lost within 10 min in each cell line. The rate constant for loss of platinum from this rapidly effluxing pool, measured after a 10-min loading period in 500 μM DDP, was 0.67±0.09 s−1 in sensitive cells and 1.03±0.15 s−1 (a 53% increase) in resistant cells. Between 5 and 50 min of an accumulation time course in 500 μM DDP, the size of the rapidly effluxing platinum pool remained relatively constant in each cell line, with the major contribution to the increase in total platinum over time coming from growth of the slowly effluxing platinum pool. We conclude that diminished retention of platinum in the rapidly effluxing pool of resistant cells in a major determinant of decreased DDP accumulation in these cells.

Metallothionein-mediated cisplatin resistance in human ovarian carcinoma cells.

SummaryWe have determined the ability of two human ovarian carcinoma cells to over-express metallothioneins (MTs) and the subsequent effect this elevation has on DDP cytotoxicity. Cells of 2008 and COLO 316 human ovarian carcinomas that were resistant to CdCl2 were obtained by stepwise selection and chronic culture in CdCl2 and ZnCl2. The 2008/MT cells were 3.2-fold resistant to CdCl2 and 4.1-fold resistant to DDP; they had 23-fold elevated MTs. The COLO/MT cells were 1.2-fold resistant to CdCl2 and 3.3-fold resistant to DDP, and they had 9-fold elevated MTs. Glutathione (GSH) was also elevated in the Cd-resistant sublines. However, four times more intracellular thiols were contributed by the MTs than by the GSH. 2008 and 2008/MT cells were examined in more detail to elucidate the mechanism of DDP resistance. Depletion of GSH with D,L-buthionine-S,R-sulfoximine (BSO) had no effect on the sensitivity of these cells to either CdCl2 or DDP. Uptake of [195m Pt]DDP in 2008 and 2008/MT cells was identical. Fractionation of the cytosol from [195mPt]DDP-exposed cells on Sephadex G-75 revealed that 17% of the total cellular Pt in 2008/MT cells was associated with the MT fraction, as against 4% in the parent 2008 cells. This increase corresponded to a concomitant loss of Pt from the particulate fraction. Fractionation of 2008 cells selected with DDP (2008/DDP cells) indicated that elevated MTs did not contribute to the DDP resistance of these cells. Only 2% of the total cellular Pt was in the MT fraction in 2008/DDP cells. These results showed that elevation of MTs may be one mechanism of DDP resistance in ovarian carcinoma; however, in vitro selection with DDP does not trigger this mechanism.

Differential toxicity of carrier-bound methotrexate toward human lymphocytes, marrow and tumor cells.

Abstract Methotrexate that was covalently linked to poly- l -lysine (mol. wt 3,000 and 60,000) (MTX-PLL 3K and 60K) was more inhibitory to the growth of five cell lines from human solid tumors (IC 50 5−10 × 10 −8 M and 1−2.6 × 10 −8 M respectively) than to the growth of five lines of human lymphocytes (IC 50 5−8 × 10 −7 M and 2−5 × 10 −7 M). In contrast, both methotrexate that was covalently linked to human serum albumin (MTX-HSA), and the free drug, were equally toxic to the two classes of cells, with IC 50 of 3−15 × 10 −7 M and 2−7 × 10 −8 M, respectively, for the cell types. Uptake studies showed that, whereas MTX and MTX-HSA were transported equally well into WI-L2 lymphocytes, human bone marrow cells, and an astrocytoma tumor line, uptake of MTX-PLL by the astrocytoma cells at 37° was three to four times greater than uptake by WI-L2 lymphocytes or marrow cells. [ 3 H]Deoxyuridine ([ 3 H]-Urd) incorporation studies indicated that low concentrations of MTX-PLL 60K (5 × 10 −7 M) resulted in inhibition of the target enzyme dihydrofolate reductase (DHFR) in the astrocytoma cells, but no iinhibition of DHFR occurred in WI-L2 lymphocytes or marrow cells until concentrations were reached where the carrier itself became toxic (5 × 10 −6 M). Two inhibitors of the lysosomal enzymes, chloroquine and lupuptin, were able to reverse the toxicity of MTX-PLL 60K against the astrocytoma cell line, increasing its IC 50 from 2 × 10 −8 to 2 × 10 −7 M. Both lysosomal inhibitors had no effect on the toxicity of MTX-PLL 60K against the WI-L2 lymphocytes or of MTX or MTX-HSA against either cell type, indicating that the increased toxicity of MTX-PLL 60K against the tumor cells was due, in part, to the ability of the lysosomes of these cells to convert MTX-PLL 60K either to the free drug or to a derivative that was effective in inactivating DHFR. These results suggest that comparable differential toxicity between marrow and tumor cells might also be achieved in vivo if MTX-PLL is infused over long periods at a rate that would maintain a constant serum concentration sufficient to kill tumor cells without affecting bone marrow cells.

Regulation of HSP60 mRNA expression in a human ovarian carcinoma cell line

The expression of the 60-kDa heat-shock protein (HSP60) varies markedly among patients with ovarian carcinoma, and high-level expression predicts poor survival in such patients treated with cisplatin (DDP)-containing chemotherapy programs. We investigated the expression of HSP60 in human ovarian carcinoma 2008 cells and an 11-fold DDP-resistant subline 2008/C13*5.25. Heating for 2 h at 44°C produced a 2.7±0.16-fold increase (mean ± SD) that was maximal at 4 h after the start of heat exposure. Exposure to an IC50 concentration of DDP for 1 h induced a 1.8±0.03-fold increase inhsp60 expression. The opposite was true for cadmium and zinc, both of which induced increases in metallothionein IIA but not in thehsp60 message. 2008/C13*5.25 cells constitutively over-expressedhsp60 mRNA by 1.7±0.16 orders of magnitude and contained a 3.8±0.45-fold higher level of HSP60 as detected by immunocytochemical staining. 2008/C13*5.25 cells showed 1.2-fold cross-resistance to thermal killing. Expression ofhsp60 was markedly reduced in 2008 xenografts as compared with 2008 cells growing in vitro; however, neither serum starvation nor refeeding altered the message level. Exposure to a variety of growth factors and drug treatmens known to alter the DDP sensitivity of 2008 cells, including epidermal growth factor, 12-O-tetradecanoylphorbol-13-acetate, buthionine sulfoximine, ouabain, and forskolin, did not alterhsp60 expression. These results suggest a role for HSP60 in mediating resistance to both DDP and hyperthermia but indicate that thehsp60 mRNA levels are not regulated by the factor listed above.

Modulation of the peritoneal clearance of liposomal cytosine arabinoside by blank liposomes

SummaryLiposomes containing cytosine arabinoside (ara-C) release drug slowly and can be used to maintain a locally high concentration of ara-C in the peritoneal cavity for intracavitary chemotherapy. However, a significant amount of active drug does reach the systemic circulation and contributes to systemic toxicity. We have devised a novel method of decreasing toxicity and increasing intraperitoneal half-life by pretreatment with “blank” liposomes containing no active drug. This technique has resulted in prolongation of intraperitoneal half-life of the liposomal ara-C from 21 h to 165 h, enabling maintenance of a therapeutic drug concentration even at 11 days after initial injection. One hundred percent cures (60-day survival) were achieved with a single-dose therapy begun 1 day after i. p. implantation of 106 L1210 leukemia cells.

Analysis of the cytotoxic interaction between cisplatin and hyperthermia in a human ovarian carcinoma cell line.

Expression of the heat-shock protein HSP-60 is associated with poor survival in patients with ovarian carcinoma. We examined both the nature of the interaction between hyperthermia and cisplatin (DDP) using the human ovarian carcinoma cell line 2008 and the effect on this interaction of the induction of the heat-shock response. The nature of the interaction was assessed using medianeffect analysis. Despite the observation that 45°C hyperthermia increased the intracellular uptake of the DDP analog [3H]-cis-dichloro(ethylenediamine)platinum(II)(DEP) during a 1-h exposure by 155%±5% (P=0.02), median-effect analysis indicated only cytotoxic additivity (combination index at the level of 50% cell kill, 0.96±0.25). When cells were first exposed to hyperthermia for various periods and then allowed to incubate at 37° C for 4 h to allow induction of the heat-shock genes before being treated with DDP for 1 h, there was a very small degree of antagonism between hyperthermia and DDP (combination index at 50% cell kill, 1.11±0.04). Our results indicate that DDP and hyperthermia interact only in an additive manner against this human ovarian carcinoma cell line and that the induction of heat-shock proteins by hyperthermia does not significantly antagonize the activity of DDP.

THE APPLICATION OF SPLINE FUNCTIONS TO THE PHARMACOKINETIC ANALYSIS OF METHOTREXATE INFUSED INTO MALIGNANT EFFUSIONS11Supported by National Cancer Institute Grants CA-23100 and CA-23334, National Science Foundation Grants MCS-7901800 and MCS-7906228, and National Institutes of Health Grant CA-26666.

Publisher Summary This chapter describes the application of spline functions to the pharmacokinetic analysis of methotrexate infused into malignant effusions. It discusses how spline functions can be used in smoothing intrinsically variable data arising from pharmacokinetic experiments. This technique can be especially useful in the initial or exploratory stages of the analyses of such data and can be helpful in ascertaining the suitability of tentatively held models. By providing continuous values of the concentrations and their derivatives through time, the plausibility of a first order linear kinetic model can be examined. A particularly convenient representation for spline functions with a given breakpoint sequence is made by the B-spline basis.