Effects of cell density on drug-induced cell kill kinetics in vitro (inoculum effect)

Ohnuma, Takao; Arkin, Hadara; Holland, James F.

doi:10.1038/bjc.1986.192

Cited by 40 publications

(25 citation statements)

References 17 publications

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“…We introduced a combination of these 2 agents, termed A & P (adriamycin and platinum) (Vogl et al, 1976), which has become the basis, by adding more drugs, for the treatment of patients with ovarian cancer, small cell lung cancer and other neoplasms. Recently, we have demonstrated that DXR has a positive inoculum effect in vitro and loses efficacy at high cell densities, whereas DDP does not (Ohnuma et al, 1986). Moreover, we have shown that DXR penetrates poorly into the core of multicellular tumour spheroids (MTS) whereas DDP shows no such penetration gradient (Inoue et al, 1985).…”

mentioning

confidence: 99%

Interactions of doxorubicin and cis-platin in squamous carcinoma cells in culture

Kohno

Ohnuma²,

Kaneko³

et al. 1988

Br J Cancer

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Summary Doxorubicin (DXR) has a positive inoculum effect and penetrates poorly into the core of multicellular tumour spheroids (MTS). Cis-platin (DDP) displays neither of these characteristics. We evaluated whether combining these 2 agents would influence the cell kill effect at a tumour mass level. MTS were produced from a PC-10 squamous lung carcinoma cell line. MTS were exposed to either drug first for h with different intervals between exposure. Cells were then trypsinized to a single cell suspension and subjected to clonogenic assay. Combination effects were analyzed by median effect plot analysis. The more MTS ml-' medium, the lower the cell kill effect of DXR. Simultaneous exposure to the 2 drugs was synergistic. DXR exposure first followed by DDP was less efficacious than, or the same as, the simultaneous exposure. In contrast, DDP followed by DXR was more efficacious with the best cell kill at a 1 h interval between each drug. This phenomenon was observed even at non-toxic doses of DDP. The fluorescent microscopic study of DXR indicated that prior exposure of MTS to DDP resulted in increased DXR penetration into the MTS core leading to heightened synergism with this sequence. These data suggest that the proper combination of DXR plus DDP should be in sequence with DDP first. Clinical, toxicological and pharmacological trials of DDP administration first, followed by DXR, are warranted.Doxorubicin (DXR) and cis-platin (DDP) are both highly active anticancer agents widely used in the treatment of human cancer. We introduced a combination of these 2 agents, termed A & P (adriamycin and platinum) (Vogl et al., 1976), which has become the basis, by adding more drugs, for the treatment of patients with ovarian cancer, small cell lung cancer and other neoplasms. Recently, we have demonstrated that DXR has a positive inoculum effect in vitro and loses efficacy at high cell densities, whereas DDP does not (Ohnuma et al., 1986). Moreover, we have shown that DXR penetrates poorly into the core of multicellular tumour spheroids (MTS) whereas DDP shows no such penetration gradient (Inoue et al., 1985). These pharmacological differences at the tumour mass level indicate that DXR should be best used when tumour size is small and cell density low. Therefore, we hypothesized that the proper sequence of A & P should be DDP first, followed by DXR, rather than vice versa. This communication sets forth the experimental proof of our hypothesis and elucidates a possible mechanism for this effect. Materials and methodsHuman tumour cell line PC-10 squamous lung carcinoma cell line was used in these experiments (Kinjo et al., 1979). Cells were maintained as a monolayer in RPMI-1640 medium (GIBCO, Grand Island, NY), supplemented with 10% (v/v) heat-inactivated foetal bovine serum (FBS) (Gibco) at 37"C in a 5% CO2/95% humidified air atmosphere. These cells were subcultured after trypsinization with 0.2% trypsin (Type III from Conditions of drug exposure and determination of cell survival After 3 weeks of culture, MTS with a diame...

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confidence: 99%

Interactions of doxorubicin and cis-platin in squamous carcinoma cells in culture

Kohno

Ohnuma²,

Kaneko³

et al. 1988

Br J Cancer

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“…Similar effect was described also for vincristine (5). In contrast, cisplatin and carboplatin do not exhibit the inoculum effect, since cell density does not affect cytotoxic doseresponse curves and cellular accumulation of these drugs (4,25).…”

Section: Discussionmentioning

confidence: 48%

“…Both the flush time and the laser warm-up time were set to 3 sec. The ion signal of two copper isotopes, 63 Cu and 65 Cu, was monitored to reveal possible polyatomic interferences of 40 Ar 25 Mg, 40 Ar 23 Na etc. Signal of the most abundant isotope, 63 Cu was used for data evaluation.…”

Section: Dna Fragmentation Analysismentioning

confidence: 99%

Copper ions regulate cytotoxicity of disulfiram to myeloid leukemia cells

Navrátilová

Jungová²,

Vaňhara³

et al. 2009

Int J Mol Med

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Abstract. White blood cell (WBC) count is considered a prognostic risk factor in acute myeloid leukemia. As density of leukemic cells increases, the cytotoxic activity of certain anticancer drugs, such as vincristine and doxorubicin, progressively decreases. In this study, we investigated the cell density-dependent induction of apoptosis of human acute myeloid leukemia U937 and ML-1 cells by disulfiram (DSF), the dithiocarbamate drug recently proposed for treatment of human cancers. This effect is dependent on uptake of extracellular copper and its intracellular accumulation. Highdensity cells cannot uptake and accumulate this metal to a sufficient level that would allow induction of apoptosis due to progressive decrease of its extracellular concentration. Simple addition of copper can resume sensitivity of highdensity leukemic cells to DSF and improve efficiency of antileukemic therapies using this drug, thus providing benefit to patients with high WBC count.

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“…ETO was kindly provided by Drs (Muler et al, 1992). No inoculum effect was observed with either parental or seled cells (Ohnuma et al, 1986). Each cell lne was characterised by the concentration of drug resulting in 10% survival (D1O) and by its resistance index (RI), defined as the ratio of the D0 value for a particular subline over that for the parental cell line.…”

Section: S Soul Eta 490mentioning

confidence: 99%

Mechanisms of resistance to combinations of vincristine, etoposide and doxorubicin in Chinese hamster ovary cells

Souès

Laval²,

Charcosset³

1995

Br J Cancer

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Summary We have isolated from Chinese hamster ovary cells, 30 sublines resistant to vincristine, doxorubicin or etoposide and 43 sublines evading treatment with a pair of these drugs. Isolated in one step and under low selective pressure. subhnes were 3-to 25-fold more resistant to their selecting drug(s) than the parental cells. Possible P-glycoprotein-associated multidrug resistance was investigated through pgp gene copy number and mRNA expression level. DNA topoisomerase II alteration was evaluated from the ability of nuclear extracts to form cleavable complexes. Vincristine (all sublines) and doxorubicin (6 7 sublines) preferentially selected for pgp gene amplification and mRNA overexpression, whereas selection with etoposide resulted in a decrease of cleavable complex formation in 11 out of 13 sublines. A common pgp gene-mediated resistance was found in the 13 doxorubicin plus vincristine-selected sublines, whereas all but one of the 12 etoposide plus vincristineresistant sublines displayed both pgp mRNA overexpression and decreased ability to form cleavable complexes. Among the 18 doxorubicin plus etoposide selected sublines, five exhibited a decreased ability to form cleavable complexes only, six exhibited pgp mRNA overexpression only and six exhibited both alterations. Overall, drug resistance could not be attributed to either mechanism in three of the 73 sublines. We conclude that under low selective pressure it is possible to find a combination of drugs which require simultaneous selection of more than one resistance mechanism; such cells emerge with very low frequency.

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Effects of cell density on drug-induced cell kill kinetics in vitro (inoculum effect)

Cited by 40 publications

References 17 publications

Interactions of doxorubicin and cis-platin in squamous carcinoma cells in culture

Interactions of doxorubicin and cis-platin in squamous carcinoma cells in culture

Copper ions regulate cytotoxicity of disulfiram to myeloid leukemia cells

Mechanisms of resistance to combinations of vincristine, etoposide and doxorubicin in Chinese hamster ovary cells

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