Toxicity of the mitochondrial poison dequalinium chloride in a murine model system.

Dequalinium chloride (DECA), a cationic, lipophilic mitochondrial poison, selectively targets the mitochondrial membrane of certain epithelial carcinoma cells, in which it inhibits cellular energy production. It has demonstrated potency as a cytotoxic agent specific for carcinomas and may provide a novel approach for cancer therapy, either as a single agent or as an adjunct to conventional chemotherapy. The purpose of this study was to determine the toxicity of DECA in the murine model. One hundred female BALB/c mice were divided into three schedule groups. Group one received a single intraperitoneal (ip) dose of DECA at 10, 15, 20, or 25 mg/kg of body weight. Group two received DECA at 6, 7, 8, 9, or 10 mg/kg ip every other day (QOD), and group three received DECA at 10, 11, 12, 13, or 14 mg/kg ip every 7 days. Over a 30- to 60-day period, acute and subchronic toxicities were evaluated on the basis of the following clinical parameters: respiratory distress, weight loss, and mortality. After a single ip administration, we found a maximum tolerated dose of 15 mg/kg and a lethal dose (LD50) of 18.3 mg/kg. Single ip doses of 20 and 25 mg/kg produced > 50% mortality. Histologic examination of the tissues revealed significant damage to the liver and kidneys, with pulmonary congestion occurring secondary to renal-hepatic failure. A cumulative assessment revealed that 60% of the animals tolerated 15 doses of 6 and 7 mg/kg QOD and that 100% tolerated 5 doses of 11 and 12 mg/kg (every 7 days). Higher DECA doses under either regimen induced severe toxic effects and mortality.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a human ovarian carcinoma cell line: UCI 101.

A new epithelial ovarian carcinoma cell line (UCI 101) has been established from the ascitic fluids and solid tumor of a patient with progressive papillary adenocarcinoma of the ovary shown previously to be refractory to combination chemotherapy consisting of cyclophosphamide, Adriamycin, and cisplatin as well as single-agent chemotherapy of taxol and high-dose cisplatin. The UCI 101 cell line grows well with an in vitro doubling time of 24 hr. The cell line expresses the B 72.3 (Tag 72), CA125, MH99 (ESA), and E29 (EMA) cell surface antigens and AE1/AE3 cytokeratins. This cell line overexpresses (as determined by immunocytochemistry) both p-glycoprotein and the epidermal growth factor receptor. The in vitro drug response to single agents including Adriamycin, cisplatin, dequalinium chloride, etoposide, 5-fluorouracil, taxol, and tumor necrosis factor was examined. Intraperitoneal transplantation of the cells into athymic mice resulted in foci of tumor on all peritoneal surfaces including the viscera and diaphragm ultimately leading to solid bulky disease with massive production of ascites. High levels of CA125 (> 500 units/ml) were detected in the serum of tumor-bearing mice. Cytogenetic analysis of cultured cells shows several marker chromosomes containing deletions, duplications, and translocations. Cytologic and histologic evaluation of the xenograft revealed morphological characteristics identical to those of the original tumor.

Characterization of normal human brain cultures. Evidence for the outgrowth of leptomeningeal cells.

To establish the histogenetic identity of the predominant cell type in monolayer cultures of normal human adult brain, eight brain specimens were placed into culture and characterized according to cell kinetics, karyotype, antigenic expression, and ultrastructural features. The protein profiles of both the cell layer and the medium were analyzed in selected cultures using sodium dodecyl sulfate polyacrylamide gel electrophoresis and diethylaminoethyl cellulose chromatography. All cultures displayed a limited life span in vitro; marked contact inhibition at confluence; a normal karyotype; an intracytoplasmic and extracellular glycoprotein profile consisting of fibronectin, procollagen type III, laminin, and collagen type IV; specialized intercellular junctions; and interstitial collagen chain synthesis. All of these features were identified in our previous study of human leptomeningeal cultures. The results of immunocytochemical staining for glial fibrillary acidic protein were negative in all cultures of normal human brain, except in early passages in two cultures, which lost the glial cell marker during subsequent passages; immunostains for vimentin were positive in all cells in all cultures. These results support the hypothesis that, in this study, cultures derived from normal human brain are not of glial origin. Our findings also suggest that glial cells are less well-suited to monolayer growth under our culture conditions than are other cell types in enzyme-dissociated brain tissue placed in culture, especially leptomeningeal cells. The identification of leptomeningeal cells as the predominant cell type in normal human brain cultures may prove useful in attempts to foster the growth of human glial cells by culturing brain samples under conditions that prohibit the growth of leptomeningeal cells. Under such conditions, astrocytes, oligodendroglia, and ependymal cells could be isolated with greater ease and cultured separately. These purified cultures of different glial cell types would then provide a more relevant in vitro model for studying human neurological diseases.

Development of latent residual drug damage to the hematopoietic marrow during the subsequent growth of tumors.

Growth of the Lewis lung (LLca) tumor in BDF1 mice was found to be accompanied by a marked expansion of the multipotential stem (CFUs-8) and committed erythroid (BFUe) and myeloid (CFU-gm) progenitor cells of the marrow with a concomitant depression of more differentiated compartments. The long-term effects of adriamycin (AdR), busulfan (BU), cis-diaminedichloroplatinum II (DDP), and 5-fluorouracil (5-FU) on the LLca-induced expansion of the CFUs-8 and CFU-gm were investigated at eight weeks after drug treatment. Of the four drugs studied, only BU demonstrated a reduction of CFUs-8 at eight weeks after treatment and prior to tumor inoculation. However, all of the drugs were found to prevent the expansion of the CFUs-8 compartment after 16 days of tumor growth. BU also resulted in a depressed CFU-gm compartment at the time of tumor inoculation, while CFU-gm in ADR-, DDP-, and 5-FU-treated animals was either at control levels (AdR), or unexpectedly elevated (DDP and 5-FU). Similar to the observations made for CFUs-8, all drugs prevented the expansion of the CFU-gm associated with tumor growth. The data suggest that qualitative differences observed between the long-term effects of the drugs on the marrow compartments may be more accurately related to the temporal "fixation" of residual drug damage brought about by enhanced differentiation of a drug-limited pluripotential CFUs, than to the actual magnitude of hematopoietic damage.

Residual adriamycin (AdR)-induced hematopoietic damage: a consideration for subsequent radiotherapy.

The long-term effect of adriamycin (AdR) on the radiation response of hematopoietic marrow was studied at 16 weeks after treatment with a MTD (10 mg/kg) for the BDF1 mouse. The radiation response was monitored in both the "stem cell" (CFUs-8) and myeloid (CFU-gm, granulocyte, WBC) compartments, as well as the erythroid (BFUe, CFUe, HcT) compartments of the marrow for 14 days following a whole body dose (TBI) of 4.5 Gy X ray. At the time of irradiation, animal and spleen weight of AdR treated animals were reduced while HcT and WBC remained at control levels. At the same time the granulocyte and CFUs-8d compartments were depressed, while the BFUe compartment was expanded. The CFUe and CFU-gm compartments remained at control levels. For all marrow compartments, treatment with AdR 16 weeks prior to 4.5 Gy resulted in a radiation response deficit determined from the temporal recovery curves. The data suggest that manifestation of long-term AdR injury, at least through 16 weeks following treatment, is dependent on a subsequent stress of sufficient magnitude to enhance the proliferative activity associated with hematopoietic cell production and differentiation. A comparison is made between these observations and previously reported long-term drug-induced hematopoietic injury.

Haemopoietic modulation in tumour-bearing animals: enhanced progenitor-cell production in femoral marrow.

Altered haematopoiesis in the femoral marrow was observed in mice bearing the Lewis lung carcinoma (LLca). During tumour growth, a marked reduction was observed in the myeloperoxidase-positive cells (granulocytes) of the marrow 7 days after inoculation of the LLca tumour reaching a nadir (17% of control) by day 28. Accompanying this suppression of mature white cells was a gradual expansion of the CFUc-GM compartment followed by an increase in the number of femoral CFUs. Humoral-stimulating activity (HSA) increased through day 14 in the serum of these animals; then returned to control levels by day 28. During this same interval, the more primitive erythroid progenitor (BFUe) compartment expanded to 168% of control, while the more differentiated (CFUe) compartment was reduced (45% of control at day 28). Reductions in both 59Fe-incorporation and erythroblasts/femur confirmed the suppression of erythroid differentiation in marrow during tumour growth. Similar results were observed following the daily injection (188 mg equivalent dose; q 24 hr X 10) of the supernatant prepared from LLca tissue. Marked differences were observed between the response of the spleen and the marrow to the supernatant. The data suggest that the growth of the LLca tumour results in a dissociation of the normal continuity of haematopoietic steady-state differentiation in the marrow of tumour-bearing animals.

Effect of prolonged expansion of marrow progenitor compartments following doxorubicin (ADR) on subsequent radiation tolerance.

The long-term effects of a maximum tolerated dose of doxorubicin (ADR) (10 mg/kg, LD10/60) on the recovery of the hematopoietic compartments of the femoral marrow from radiation (450 rad) were investigated over a 32-week interval using a mouse model. Comparative radiation response curves, estimating hematopoietic proliferative potential, were used to establish response deficits (RD) for individual compartments of ADR-treated marrow. The RD data suggest that two potentially discrete lesions result from ADR treatment: one lesion associated with acute toxicity and a second developing 8-16 weeks after drug treatment. A depletion of the older hematopoietic stem cell (CFU-S8d) compartment was observed to accompany the development of the second lesion. Data are presented suggesting that both initial stem cell kill, as well as an accelerated aging of the CFU-S8d by prolonged expansion of progenitors, eventually lead to a depletion of stem cell reserve manifested by a progressive loss of radiotolerance with time.

Enhancement of colony-stimulating activity (CSA) on murine CFU-gm by the H-4-II-E2 (H4) tumor cell line of the rat.

The effect of the H-4-II-E2 (H4) rat tumor cell line on murine granulocyte/macrophage colony-forming units (CFU-gm) was studied in vitro using a bilayer (agar/methylcellulose) culture system over the tumor cell feeder and 10% colony-stimulating activity (CSA). The H4 cells demonstrated an amplification of CSA from several sources and of CFU-gm growth of murine marrow, including the CSA present in L-cell-conditioned medium (L-CSA; 200% of control). The amplification did not result from CSA produced by the H4 cell line, nor was cell-to-cell contact necessary for enhanced CFU-gm growth. Amplification of L-CSA was not mediated by endogenous or exogenous prostaglandin E concentrations in the in vitro system. Furthermore, incubation of the non-adherent marrow cell population with H4 tumor cells for 24 h prior to assaying for CFU-gm resulted in more colonies, independent of the continued presence of H4 tumor cells. The data suggest that the H4 tumor cells produce a readily diffusable, soluble factor that may amplify the effect of L-CSA on CFU-gm by stimulating a more primitive progenitor cell that expands the CFU-gm population.

The fusion of erythrocytes by treatment with proteolytic enzymes and polyethylene glycol.

Polyethylene glycol (PEG) has been utilized to induce homokaryocyte formation in avian and mammalian erythrocytes previously treated with proteolytic enzymes. PEG of molecular weight 6,000-7,5000 was found superior to 1,500 and 20,000 MW PEG. Cells exposed to protease alone, prior to PEG treatment, fused to a high degree (60-95% multinucleated cells), whereas trypsin or pepsin treatment alone allowed very little fusion (2.5%). Trypsin lowered the effectiveness of protease when used in combination. Cells which were not treated with proteolytic enzymes agglutinated in the presence of PEG but did not fuse to a significant extent (0.01%). Fusion was also markedly dependent upon the rate at which PEG was eluted during the fusion process. Electron microscopy indicated that fusion began during the elution of PEG from the agglutinated cells.