Roland A. Pattillo

Skin ulcers due to adriamycin

Local skin necrosis at the site of intravenous or intra‐arterial adriamycin infusion is an infrequent, but serious complication. Ulcers secondary to adriamycin have insidious beginnings, but progress to a much deeper extent than would be expected from their initial appearance. Deep structures, such as tendon or bone, may become exposed. The ulcers are indolent and do not develop a granulation tissue response or epithelialization, as might be expected from their early appearance. Injections of adriamycin in the dorsum of the hand should be avoided when possible, since tendons have little skin cover and the area is difficult to cover with local tissue if there is skin loss. While prevention is important, early surgical treatment may prevent progressive deep involvement and seems warranted when the patient has a life expectancy of months or years. Wide excision of all inflamed tissue is the treatment of choice, with split‐thickness skin grafting or flap coverage.

THE HORMONE‐SYNTHESIZING TROPHOBLASTIC CELL IN VITRO: A MODEL FOR CANCER RESEARCH AND PLACENTAL HORMONE SYNTHESIS

The human trophoblast, the hormonally functional cell of the placenta, differentiates in the first-order cleavage of the fertilized human ovum immediately following conception.1 Hormonal function from the placental trophoblast can be detected in maternal serum by human chorionic gonadotropin (HCG)2 and human placental lactogen (HPL) assays shortly following the 58-cell stage of the human ovum. Thus, trophoblastic cellular differentiation and placental hormone synthesis are among the earliest morphological and biochemical differentiations to occur in the human. This cell type has not been established in vitro. Choriocarcinoma is a unique malignancy of trophoblastic cells which follows normal pregnancy, spontaneous abortion, or hydatiform mole. This tumor closely parallels the normal placenta in hormone function and cellular metabolism, with the exception that the limited invasiveness of the normal placenta during its implantation is far exceeded by that of the malignant tumor, which, proceeding unrestricted, culminates in the death of the patient within a period of less than 1 year.4 A morphological similarity can be observed among the trophoblastic cells of the human blastocyst less than 92 hours after conception (FIGURE l ) , the invading normal placenta of the first trimester (FIGURE 2), and the rapidly proliferating choriocarcinoma (FIGURE 3 ) .

Radioimmunoassay of free ß-subunit of human chorionic gonadotropin in diagnosis of high-risk and low-risk gestational trophoblastic disease

Summary A radioimmunoassay was performed with monoclonal antibody 1E5, which distinguishes free s-subunit of human chorionic gonadotropin in the presence of intact human chorionic gonadotropin. Serum samples were obtained from 68 pregnant women, 9 with hydatidiform mole who underwent spontaneous remission, 12 with hydatidiform mole who developed gestational trophoblastic disease, 5 with metastatic gestational trophoblastic disease of high-risk category, and 1 with choriocarcinoma concomitant with pregnancy. The concentrations of free B-subunit of human chorionic gonadotropin and total s-subunit were determined on the sera. The assay data were expressed as a ratio of nanograms of free s-subunit per 1000 mlU of total s-subunit. The ratios, analyzed by the Wilcoxon two-sample test, indicated a highly significant correlation between high ratios and the eventual diagnosis of high-risk gestational trophoblastic disease (p = 0.0019). This study suggests that the excessive production

CONTROL MECHANISMS FOR GONADOTROPHIC HORMONE PRODUCTION IN VITRO

SummaryAn internal control mechanism capable of controlling gonadotrophic hormone secretion in the human trophoblast has been identified by incubation of an established cell line with a precursor of progesterone.Biological, biochemical, and electron microscopic findings verify inhibition of gonadotrophic hormone synthesis, depletion of glycogen, activation of phosphorylase, the glycogen-metabolizing enzyme, and ultrastructural appearance of liquid-containing cytoplasmic granules in single cells, indicative of steroid synthesis.

Effect of cyclic 3':5'-AMP derivatives prostaglandins and related agents on human chorionic gonadotropin secretion in human malignant trophoblast in culture.

SummaryThe secretion of human chorionic gonadotropin (hCG) is stimulated by addition of N6, O2′-dibutyryl cyclic 3′:5′-AMP (dbcAMP) or theophylline to normal term placenta and human malignant trophoblast cells in vitro. To understand better the specificity of this process. malignant trophoblast cultures were incubated with 3′:5′-cyclic AMP (cAMP) derivatives, prostaglandins and other agents for 1 to 3 days, and the secretion of radioimmuno-assayable hCG was measured. Whereas dbcAMP was the most potent agent in stimulating secretio of hCG, the N6- and O2′-monobutyryl derivatives of cAMP and phosphodiesterase inhibitors (theophylline, papaverine, 3-isobutyl-1-methylxanthine) also increased the secretion of the hormone. A slight increase in hCG secretion was observed following addition of adenine. By contrast, butyrate, cAMP, cyclic 3′:5′-GMP (cGMP), dbcBMP, 5′-AMP, adenosine, L-epinephrine and prostaglandins E1, E2, F1α and F2α were ineffective. Particulate fractions from sonicates of malignant trophoblast cultures contained adenylate cyclase activity which was stimulated more than 10-fold by NaF, but not by either catecholamines or prostaglandins. The relatively specific stimulation of hCG secretion suggested that a regulatory process involving cAMP may have physiological significance in the trophoblast.

Effects of methotrexate on established cell lines of human choriocarcinoma

Abstract Cell lines of human choriocarcinoma (BeWo and Jar lines) were incubated in vitro for up to 7 days with 0·10, 10 , or 1000 μM methotrexate (MTX). The cell population did not change significantly for up to 6 days after daily addition of 0·10 to 1000 μM MTX to the culture medium, that was also changed each day. During the same time period the cell population quadrupled in untreated control flasks. The drug caused an increase in amount of protein per cell and in the protein: DNA ratio. Incubation for 5 hr with MTX at concentrations of 0·10 and 10 μM resulted in 83 and 93% inhibition, respectively, of deoxyuridine- 6 - 3 H incorporation into DNA. After 3 days of incubation with either 0·10 or 10 μM MTX, no incorporation of labeled deoxyuridine into DNA was observed. Incubation in the presence of the antifolate resulted in a rapid dose-dependent compensatory increase in incorporation of thymidine- 2 - 14 C into DNA, rising to 135 and 197% of untreated control levels after 24 hr of incubation with 0·10 and 10 μM MTX, respectively. At later times a decrease occurred in absolute rate of thymidine incorporation into DNA, but not in the relative rate compared to untreated cells of the same age. MTX did not significantly alter the incorporation of leucine- 3 H or uridine- 3 H into the trichloroacetic acid precipitate, nor of glucose- 3 H into DNA, RNA, protein, or lipid in the trophoblastic cells. Omission of serum from the daily medium did not change the above results. Unsuccessful attempts were made to subculture cells previously incubated with concentrations of 0·10 μM MTX or greater, although successful subculturing was possible in the presence of 0·01 μM MTX. The deleterious effects of MTX observed in both the BeWo and Jar cell lines were consistent with the suggestion that the major effect of the drug is to inhibit dihydrofolate reductase.

Early Stimulation of Human Chorionic Gonadotropin Secretion by Dibutyryl Cyclic AMP and Theophylline in Human Malignant Trophoblast Cells in vitro: Inhibition by Actinomycin D, α-Amanitin, and Cordycepin

Human malignant trophoblast cells (BeWo line) in culture were employed to investigate the early stimulation of human chorionic gonadotropin (hCG) secretion by 1 mM dibutyryl cyclic AMP and 1 mM theophylline (dbT). The earliest increase in secreted immunoreactive hCG occurred at 3 1/2 h following addition of dbT, and was preceded by an increase in intracellular hCG. These results suggested that dbT stimulated hCG synthesis, rather than release. Addition of either 0.062 mug/ml actinomycin D or 100 mug/ml cordycepin along with dbT, or 3 mug/ml alpha-amanitin 9 h prior to addition of dbT, prevented the increase in hCG secretion at 3 1/2-8 h. It was concluded that synthesis of RNA (possibly messenger RNA) is required for the early stimulation of hCG secretion by dbT.

Glycogen metabolism in human hormone-producing trophoblastic cells in continuous culture

SummaryGlycogen metabolism was studied in human hormone-producing trophoblastic cells (BeWo line). Cells supplemented daily with high glucose (3 g per liter in medium) contained 5.5% glycogen and utilized glucose at an initial rate of 12.2 mμmoles per min per mg of protein. In cells supplemented daily with low glucose (1 g per liter), the initial rate of glucose consumption was 23 mμmoles per min per mg of protein and the glycogen content reached only 0.4% of wet weight 24 hr after medium replenishment. When glycogen-depleted cultures were refed glucose, an accumulation of glycogen was observed, with initial deposition occurring in areas near the cell surface. After exhaustion of extracellular glucose, cytoplasmic glycogen was utilized at a rate of 2.8 mμmoles per min per mg of protein. Addition of either low or high glucose to glycogen-depleted cells resulted in the same rate of glycogen synthesis (approximately 8 mμmoles per min per mg of protein). It was suggested that unique regulatory mechanisms function in the control of glycogen metabolism in glycoprotein hormone-producing cytotrophoblastic cells.

Immunodiagnosis in ovarian cancer: Blocking factor activity☆☆☆

Tumor immunology studies have been utilized for development of a blocking factor assay for therapy monitoring in ovarian cancer. The blocking factor index was defined as the arithmetic difference between assays conducted in the presence and absence of the patients serum compared to incubations with normal control lymphocytes. Eighteen advanced ovarian epithelial malignancies have shown blocking factor activity during treatment. Blocking factor has abated in eight patients whose clinical disease completely regressed. Chemotherapy was discontinued after 18 to 24 months. In 10 patients, blocking factor persisted and chemotherapy has been continued. Some of these patients showed decreasing blocking factor; others have shown increases, which led to death due to disseminated disease in four cases. Blocking factor activity was found to correlate with tumor growth.

Production of Tumor-Associated Antigen, TA-4, by the CaSki Cervical Carcinoma Cell Line

Previous Japanese studies described the purification of TA-4 from homogenates of tumor tissues excised from squamous cell carcinomas of the uterine cervix, and the development of a radioimmunoassay to detect TA-4 in sera of patients with this disease. The aim of the present investigation was to determine if TA-4 was produced by the CaSki cell line, established in culture ten years ago from epidermoid carcinoma of the uterine cervix. The radioimmunoassay detected the TA-4 antigen in the CaSki cells, but not in cell lines derived from either choriocarcinoma or breast carcinoma. The TA-4 concentration in the CaSki cell lysate exceeded that in the CaSki culture fluid by more than twentyfold, and exceeded the concentration of human chorionic gonadotropin beta-like immunoreactive material by nearly two orders of magnitude. Antiserum to TA-4 was used to immunoprecipitate biosynthetically labeled TA-4 from CaSki cultures that had been incubated with [3H]leucine. After electrophoresis and autoradiography, the immunoprecipitated material showed a major band corresponding in apparent molecular weight (48,000 daltons) to TA-4 originally isolated from squamous cell carcinoma tumors. It is concluded that the CaSki cell line constitutes an ideal model with which to investigate the biosynthesis and regulation of TA-4, and a source for large-scale production of TA-4 for characterization studies as well as development of clinical diagnostic reagents.