B.U. Sevin

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.

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.

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.

Cell kinetic perturbations after irradiation and caffeine in the BG-1 ovarian carcinoma cell line

The treatment of ovarian carcinoma includes maximum surgical removal of the tumor tissue followed by irradiation or chemotherapy. In this study, the effects of caffeine on cell cycle traverse have been studied over a 168-hr period after X irradiation in BG-1 cells, an ovarian carcinoma cell line. The results were obtained with dual-parameter flow cytometric measurements of DNA and nuclear protein, using propidium iodide and fluorescein isothiocyanate. After radiation alone, a dose-related arrest of cells in G2 phase and cell kill were observed. Irradiating BG-1 cells with 5 Gy produced an accumulation of the cells in G2 at 24-72 hr postirradiation. When G2 was divided into low nuclear protein (G2A) and high nuclear protein (G2B) compartments, there was a G2A peak accumulation at 24 hr and a G2B peak accumulation at 48-72 hr. The addition of 1 mM caffeine to the culture media, starting immediately postirradiation, prevented G2 arrest, promoting a rapid traverse of cells through G2A to G2B to G1, which was associated with diminished survival.

Long-term survival in stage III and IV ovarian cancer

SummaryA total of 104 unselected, previously untreated patients with invasive stage III or IV ovarian cancer were operated on between 1977 and 1984. Nine patients were lost in follow-up, three died from non-malignant disease. Thirteen of the 92 eligible patients (15%) were observed to survive 5 years or longer. In the 13 long-term survivors, 4 had stage IV disease, 7 positive peritoneal cytology, 3 bowel resection, and 12 residual disease <2 cm after primary surgery. Retroperitoneal lymph nodes were involved in 6/9 cases. The majority of 5-year survivors (69%) received cis-platin-containing combination chemotherapy. 5/7 long-term survivors had positive second-look. At 5 years, life-quality in 9/13 patients who were free of disease, was high. It can be concluded that only patients with optimally resected stage III or IV ovarian cancer have a realistic chance of long-term survival. It is expected that increasing radicality in surgery for ovarian cancer together with platinum-based chemotherapy regimens may improve long-term survival in the future. In addition, further studies of new chemotherapeutic approaches are needed.

Prätherapeutische in-vitro-Chemosensitivität bei gynäkologischen Tumoren. Einsatz des ATP-CSA (ATP-Chemo-Sensitivitäts-Assay)

Der ATP-CSA ist eine praktikable in vitro-Testmethode zur Sensivitatstestung von Tumoren. Wie von anderen in vitro-Ansatzen bekannt, ist 4CH eine der effektivsten Substanzen beim Ovarialkarzinom. Cisplatin ist bei unbehandelten Ovarialkarzinomen wirksamer als bei vorbehandelten. Kombinationen sind wesentlich effektiver als Einzelsubstanzen. Carboplatin ist bei 20% der Ovarialkarzinomrezidive in vitro wirksam. Die Hinzunahme eine Antrazyklins beim Ovarialkarzinomrezidiv erscheint sinnvoll. Dieser Ansatz ist auch fur die Testung von neuen Substanzen und Kombinationen geeignet.

Methylxanthine verstärken die zytostatische Wirkung von Cis-Platin an gynäkologischen Krebszellen

Fur die Resistenzentwicklung gegenuber Cis-Platin (DDP) sind u.a. erhohte intrazellulare Thiolspiegel, verminderte DDP-Aufnahme in die Zelle und eine erhohte Reparaturrate nach DDP-induzierter DNS-Schadigung verantwortlich. Fur Kalzium-Kanal-Blocker wie Verapamil, Glutathion-entleerende Verbindungen und Inhibitoren der DNS-Reparatur wurde eine Modulation der zytostatischen Wirkung von Alkylantien beschrieben. Humane Ovarialkarzinom-Zellen (CAOV-3) und Zervixkarzinom-Zellen (Me-180) wurden gegenuber DDP uber 90 min und anschliesend gegenuber den beiden Methylxanthinen Pentoxifyllin (PTX) bzw. Coffein uber 24 h lang exponiert (Auswertung des ATP-Chemosensitivitats-Assays am Tag 7; Petru et al. 1990). Im Plasma maximal erreichbare DDP-Konzentrationen (2,5 (µg/ml) sowie niedrigere Konzentrationen wurden verwendet.