James P. Perras

Application of an ATP-bioluminescence assay in human tumor chemosensitivity testing

Intracellular adenosine triphosphate (ATP) is the primary energy unit of living cells, and can be quantitated by measuring the light generated with luciferase-luciferin reagent in a luminometer. The use of an ATP-bioluminescence assay, to determine tumor cell viability after exposure to chemotherapeutic agents, has been adapted to test tumor chemosensitivity in vitro. This presentation will illustrate the method of the ATP-chemosensitivity assay (ATP-CSA) using an ovarian cancer cell line NIHL:OVCAR-3 as an example and present preliminary data on 54/56 successful in vitro ATP-CSAs from 46 patients with pelvic malignancies. Fresh human tumor specimens were generally tested for single and combined drug effects at two drug concentrations (0.2 X and 1 X peak plasma concentrations). Correlation of in vitro drug sensitivity and in vivo patient response was obtained for 23 treatment regimens in 22 patients with ovarian carcinoma. The true positive rate was 100% and the true negative rate 66.7%. Our data demonstrate (a) that the ATP-CSA, measuring total cell viability, is a feasible in vitro assay for human tumor drug testing and (b) that specific criteria of in vitro chemosensitivity for this assay need to be defined by further studies, for single and combined drug exposure at different concentrations, to permit a meaningful correlation with in vivo clinical response.

In vitro potentiation of radiation cytotoxicity by recombinant interferons in cervical cancer cell lines

Background. This investigation, which evaluates the combination of radiation and interferon, bridges two clinical treatments of cancer. Radiation therapy (RT) is an integral part of cervical cancer treatment; interferons (IFN), however, are classified as modifiers of biologic response. The authors evaluated the radiation‐modulation effects of recombinant α‐IFN and β‐IFN on two different human cervical cancer cell lines: ME‐180 and SiHa. The radiation sensitivity based on the cell growth rate (logarithmic growth phase versus confluence) was also evaluated.

Comparison of paclitaxel and docetaxel (Taxotere) in gynecologic and breast cancer cell lines with the ATP–cell viability assay

The in vitro effects of paclitaxel (Tx) and docetaxel (Taxotere, Txt) are compared in this study using the adenosine triphosphate cell viability assay (ATP-CVA) in 14 cancer cell lines. Eleven cell lines were sensitive and three were partially sensitive to paclitaxel. Nine cell lines were sensitive, three were partially sensitive and two were resistant to docetaxel. Mean IC50s were 3.7-660 ng/ml paclitaxel and 5.4-540 ng/ml docetaxel. In five sensitive cancer cell lines docetaxel was more active than paclitaxel, and in six sensitive cell lines paclitaxel was more active than docetaxel on a concentration basis. Two cell lines were sensitive to paclitaxel and resistant to docetaxel. In one cell line the two compounds had similar activities. In the ATP-CVA, paclitaxel and docetaxel are very active and are partially non-cross-resistant.

Cell Cycle Perturbations of Platinum Derivatives on Two Ovarian Cancer Cell Lines

Cisplatin continues to be one of the most commonly used cytotoxic agent. Problems of drug resistance and nephrotoxicity have generated interest in new platinum derivatives. In this study, we used flow cytometry to study their effects on cell kinetics and to see if the extent of cell cycle perturbations can be used to determine relative potency. The following four platinum derivatives were tested: cisplatin, carboplatin, 254S, and NK121 on two human ovarian cancer cell lines: BG1 and CAOV3. Flow cytometric analysis revealed a dynamic spectrum of cell kinetic perturbations, which included sequential S-G2 block, concomitant S-G2 block, and a dominant S block with abolition of G2 block. Platinum derivatives NK121, 254S, and CARBO induced an average of 54.5 +/- 5.6, 21.2 +/- 5.5, and 2.5 +/- 2.8% more S-G2 blocks than cisplatin, respectively. When comparing the severity of S-G2 blocks and requiring a p-value of 0.05, the order of increasing potency was: cisplatin, carboplatin, 254S, and NK121.

Tumor heterogeneity and in vitro chemosensitivity testing in ovarian cancer.

OBJECTIVE Our purpose was to study the heterogeneity of drug response in fresh human ovarian tumors to chemotherapeutic agents in an in vitro chemosensitivity assay. STUDY DESIGN This assay evaluates total tumor cell kill by measuring the intracellular adenosine triphosphate levels of untreated controls and drug-exposed cells at various doses after culture for 6 days. The surviving fraction is calculated by dividing the treated values with the control values. One hundred tumors were tested against four single drugs (cisplatin, the active metabolite of cytoxan, 4-hydroxyperoxy-cyclophosphamide, Taxol, and carboplatin) and two drug combinations (cisplatin plus 4-hydroxyperoxy-cyclophosphamide; cisplatin plus Taxol). RESULTS There is great variation in the degree of cell death for single drugs and drug combinations among the 100 tumors tested. CONCLUSION More effective clinical response to chemotherapy may be achieved in patients with ovarian cancer by selecting the most active drugs for chemotherapy, on the basis of in vitro chemosensitivity test results for individual patients.

Chemical enhancement of cisplatin cytotoxicity in a human ovarian and cervical cancer cell line

While many advances have been made in the chemotherapy of gynecologic cancers, treatment failures remain a major clinical problem. A growing understanding of the mechanisms of tumor cell resistance to antineoplastic drugs provides a framework for the development of chemotherapy regimens containing agents capable of modulating tumor response. Using a short-term ATP bioluminescence assay we studied the ability of two methylxanthines (caffeine, pentoxifylline) and an inhibitor of ADP-ribosyl transferase (3-aminobenzamide) to enhance cisplatin cytotoxicity in gynecologic cancer cell lines. Our findings of significantly enhanced cisplatin-induced cytotoxicity with two different analysis techniques confirms the effectiveness of these agents. These results may have future clinical significance.

Characteristics of the combination paclitaxel plus doxorubicin in breast cancer cell lines analyzed with the ATP-Cell Viability Assay

SummaryPreliminary clinical data show promising activity regarding the combination of paclitaxel (TaxolTM) (TAX) and doxorubicin (AdriamycinTM) (ADR) in the treatment of breast cancer. This combination needs both further preclinical and clinical investigations to better understand the drug interaction, and to optimize the dose and schedule of these drugs. This study was done to evaluate the combination effect of TAX and ADR in three human breast cancer cell lines. The ATP-Cell-Viability Assay was used to evaluate the chemosensitivity profiles and to obtain dose response curves. For quantitation of synergism and antagonism the median-effect principle was applied and the corresponding combination index values were calculated. Drug synergism/antagonism was shown to be dose-related; synergism was enhanced at higher fractions affected. From this preclinical data, we have concluded that TAX-ADR is highly effective and partly synergisticin vitro. In spite of severe initial toxicities in early clinical trials in metastatic breast cancer patients, further clinical studies appear to be justified in order to define a tolerable dosage.

Comparison of three tumor markers—CA-125, lipid-associated sialic acid (LSA), and NB70K—In monitoring ovarian cancer

The monitoring value of three tumor markers--CA-125, lipid-associated sialic acid (LSA), and NB/70K--alone and in combination has been studied in 152 patients with invasive, epithelial ovarian cancer. The sensitivity and specificity of CA-125 (90 and 92%, respectively) were superior to those of LSA (79 and 63%, respectively) and NB/70K (76 and 74%, respectively). CA-125 by itself showed equal or higher sensitivity as well as higher specificity than in combinations with either LSA or NB/70K. CA-125 predicted cancer at second-look in 87% of cases as did LSA and NB/70K in 70 and 57%, respectively. In patients with negative second-look, CA-125 predicted the disease status in 93% as did LSA and NB/70K in 54 and 63%, respectively. On the basis of these data, the value of simultaneous determination of LSA and NB/70K, in addition to the clinically established CA-125, seems to be limited. This is due mainly to the lack of specificity of LSA and NB/70K. However, the sensitivity and specificity of CA-125 in this study were in the upper reported range.

In vitro enhancement of cis-platinum antitumor activity by caffeine and pentoxifylline in a human ovarian cell line

Cell kinetic perturbations measuring DNA and nuclear protein with dual-parameter flow cytometry were studied in vitro after cis-platinum (DDP) treatment of a DDP-resistant human ovarian cell line (BG-1). Cell viability (trypan blue exclusion) and the cells ability to replicate (replicating potential) are also reported. A dose-dependent G2 arrest and concomitant increase in cytotoxicity were observed. We also observed a marked increase in cytotoxicity of 2.5 micrograms/ml DDP with the addition of 1 mM of caffeine (CAF) or pentoxifylline (PTX). Additionally, the cells treated with DDP and CAF or DDP and PTX demonstrated a markedly diminished ability to replicate. Cell cycle perturbations, especially the G2-phase arrest, were markedly enhanced in the cells treated with DDP and CAF or DDP and PTX, when compared to that in cells treated with DDP alone. We conclude that the cytotoxicity of DDP is enhanced by CAF and PTX in vitro by inhibiting DNA repair during the S and G2 phases.

Characterization of in vitro chemosensitivity of perioperative human ovarian malignancies by adenosine triphosphate chemosensitivity assay

We report the in vitro chemosensitivity of primary and recurrent human ovarian tumor samples analyzed by adenosine triphosphate chemosensitivity assay. We defined sensitivity as a greater than or equal to 70% decrease in intracellular adenosine triphosphate versus control at 20% of the reported peak plasma concentration per agent tested. Twenty of 21 assays (95.24%) were completed successfully. Single-agent and combined dose-response patterns consisting of decreasing viability with increasing drug concentration were observed consistently. Thirteen primary tumors were assayed, with 15.4% sensitive to cisplatin, 7.7% sensitive to 4-hydroxycyclophosphamide and 53.8% sensitive to their combination. Seven recurrent tumors were assayed, with 14.3% sensitive to cisplatin, 28.6% sensitive to 5-fluorouracil, and 42.9% sensitive to their combination. Dose-response characteristics and in vitro sensitivity rates reported in this article are consistent with reports of patient response in the literature. We conclude that adenosine triphosphate chemosensitivity assay is an efficient and reliable instrument for the in vitro chemosensitivity assessment of human tumors and warrants further clinical investigation.