Selection of therapeutic agents for intraocular proliferative disease. Cell culture evaluation.

A variety of antimetabolites, steroids, and nonsteroidal anti-inflammatory agents were tested for their ability to inhibit rabbit dermal and conjunctival fibroblast proliferation in cell culture. Doxorubicin hydrochloride and fluorouracil produced notable inhibition in concentrations of less than 1 mg/L. Meclofenamate sodium and indomethacin produced notable inhibition at concentrations of 11 and 40 mg/L, respectively. Dexamethasone sodium phosphate and triamcinolone acetonide produced inhibition at 200 and 150 mg/L, respectively, but paradoxically increased proliferation almost two-fold at concentrations ranging from 1 to 30 mg/L under identical culture conditions. Methotrexate sodium demonstrated only limited effectiveness. This assay system may be a useful approach to drug selection in the treatment of a variety of ocular proliferative disorders. Fluorouracil may prove to be of significant value in the treatment of intraocular proliferative disorders.

Effect of retinoic acid on the growth and morphology of a prostatic adenocarcinoma cell line cloned for the retinoid inducibility of alkaline phosphatase.

Cells derived from the G-subline of the Dunning R-3327 rat prostatic adenocarcinoma were selected on the basis of their inducibility for alkaline phosphatase (AP) activity by retinoic acid. A p-nitrophenylphosphate-agarose overlay procedure was used to identify AP-inducible clones. The frequency of AP-inducible cells in one rapidly growing tumorigenic clone, designated 9-1C, has remained at 100% during at least 4 months of continuous culture. In culture, 9-1C cells had a mean population-doubling time in log phase of 14 hr. Retinoic acid (10 microM) did not significantly affect the rate of growth in log phase. It did, however, cause the cultures to saturate at a cell density which was 40% lower than that of control cultures. This effect on saturation density was reversible within 24 hr after removing retinoic acid from the medium. Retinoic acid-treated cells occupied greater areas on the culture dish surface, and the cross-sectional area of these cells, measured on dispersed cells by light-scatter flow cytometry, was 35 to 40% greater than that of control cells. The inducibility of 9-1C cells for AP activity decreased as the culture density increased. Cells of the 9-1C clone produced tumors when injected into male and female Fischer X Copenhagen F1 rats. No histological differences were detected between tumors grown in male and female rats. Although the tumors were poorly differentiated, primitive acinar-like structures were observed. Cells staining uniformly positive for AP activity were distributed randomly throughout the tumors. In the acinar-like structures, AP activity was localized only on the apical surfaces of the cells lining the lumens. This was also the site of enzyme activity in acini of the lateral component of the dorsolateral prostate, the source of the original R-3327 tumor. In the lateral prostatic component, AP activity was also found in the basal region of the acini, and the secretory material filling the lumens was strongly positive for the enzyme. These two regions of the tumor acini were negative for AP activity. With the exception of activity in capillaries at the basal surface, the acini of the dorsal component of the dorsolateral prostate were devoid of AP activity.

In vivo selection of androgen-insensitive cells in R3327-G rat prostate tumors: diethylstilbestrol diphosphate treatment versus orchiectomy.

Tumors grown in diethylstilbestrol diphosphate (DES)-treated rats grew significantly more slowly than tumors grown in orchiectomized animals, and tumors grown in orchiectomized animals grew significantly more slowly than tumors grown in controls (intact male rats). When these tumors (phase I) were dispersed and reimplanted into DES-treated, orchiectomized, or control rats in all possible combinations (phase II), a partial selection of androgen-insensitive cells was observed in tumors grown in DES-treated animals. Tumors grown in DES-treated phase I animals responded significantly less to DES treatment or orchiectomy in phase II. In contrast, tumors from phase I orchiectomized animals showed the same responses to orchiectomy in phase II. Since the administration of exogenous testosterone propionate prevented the growth rate inhibitory effects of both DES treatment and orchiectomy, the added effect of DES seemed to be antiandrogenic.

Experimental intraocular proliferation and neovascularization.

An experimental model of massive periretinal proliferation and intraocular neovascularization, produced in rabbits by the intravitreal injection of 250,00 cultured heterologous fibroblasts, showed no significant difference in the detachment rate (69% to 100%) or neovascularization rate (45% to 88%) between the animals injected with autologous cells and those injected with heterologous cells. Dermal fibroblasts produced a slightly higher detachment rate than conjunctival fibroblasts and were equally effective after reconstitution and subculture from liquid nitrogen storage in 7% dimethyl sulfoxide. Heterologous cells produced no clinical or histologic evidence of rejection when compared with autologous cells in the same animal and had the following advantages: (1) elimination of several biopsies and extended cell culture time; (2) a ready source of cryopreserved cells is available; (3) multiple injections of many animals can be performed within a short time; (4) in vivo and in vitro drug testing can be correlated on the same cell line.

Fluorouracil for the treatment of massive periretinal proliferation.

A single intravitreal injection of fluorouracil was effective in the treatment of an experimental model of massive periretinal proliferation. When given with an intravitreal injection of 250,000 heterologous fibroblasts, fluorouracil decreased the rate of tractional retinal detachment from 36.8% in controls (seven of 19 eyes) to 5.2% in treated animals (one of 19 eyes) at one week, and from 73.6% in controls (14 of 19 eyes) to 31.5% in treated animals (six of 19 eyes) after four weeks (P less than .05). Intraocular neovascularization was reduced from 52.6% in controls (ten of 19 eyes) to 5.2% in treated animals (one of 19 eyes) after one week and 36.8% in controls (seven of 19 eyes) to 5.2% in treated animals (one of 19 eyes) after four weeks. When supplemented by repeated 10-mg subconjunctival injections of fluorouracil, or in combination with intravitreally administered indomethacin, this effect appeared to be enhanced. Intravitreal and subconjunctival injections of fluorouracil were well tolerated and may prove to be of significant value in the treatment of human disease.

Flow cytometric analysis of RNA content in different cell populations using pyronin Y and methyl green.

Pyronin Y (PY) was used, in flow cytometric (FCM) systems, to estimate the RNA content per cell in formalin fixed EL4 leukosis tumor cells, enzyme dispersed R3327-G rat prostatic adenocarcinoma cells, mouse spleen cells stimulated with concanavalin A, and human peripheral blood lymphocytes stimulated with phytohemagglutinin. Preincubation of the cells with methyl green (MG) blocked PY binding to DNA such that the intracellular fluorescence from MG-PY was due primarily to its binding to RNA. Treatment of the cells with ribonuclease resulted in a 3- to 5-fold reduction in the fluorescence intensity of intracellular MG-PY. Mitogen stimulation of either mouse or human lymphocytes resulted in an increase in DNA (propidium iodide fluorescence) and RNA (MG-PY fluorescence) content per cell over resting levels. Further, the changes in stimulated human lymphocyte DNA and RNA contents following 24, 48, and 72 hr of cell culture were monitored. The results showed that RNA levels were significantly increased prior to that of DNA. Also, the effects of different cell cycle phase specific blocking agents on lymphocyte cell cycle traverse were investigated. We found that: a) actinomycin D inhibited the increases in cellular RNA and DNA; b) hydroxyurea inhibited the increases in cellular RNA were only slightly reduced; c) tritiated thymidine caused an accumulation of cells having high DNA and RNA contents; and d) Colcemid promoted an accumulation of cells having high DNA contents while causing a reduction of cells having high RNA contents. These results were nearly identical to reports by other investigators using the metachromatic dye acridine orange to quantitate RNA per cell. Thus, the MG-PY technique described is indicated to provide a stable and accurate measure of RNA content per cell.

Two-dimensional gel electrophoresis of membrane proteins from the R3327 prostate adenocarcinoma.

The Dunning rat prostate adenocarcinoma (R3327) is a reliable model that shares many similarities with the human tumor. Two sublines of the tumor, G and H, represent opposite extremes in histology and growth rate. Purified membrane fractions from G and H solid tumors were isolated by sucrose gradient. Tumor and normal prostate membrane proteins were labeled with 125I, incubated with G and H antisera, and precipitated by adsorption of antibody-antigen complexes to staphylococcal Protein A. Proteins were resolubilized and electrophoresed on two-dimensional gels, and the gels were autoradiographed. A total of eight labeled proteins were precipitated from the G and H tumors in the presence of G antisera. Of these, seven were homologous. One high-molecular-weight protein (Protein b) present on the G tumor was absent from the H tumor. The H tumor contained another high-molecular-weight protein (i) that was not found on the G tumor or on normal prostate. Normal prostate revealed a pattern similar to the G tumor except that Protein b appeared to be quantitatively reduced. Precipitation in the presence of H antisera showed similar patterns except that Protein b was not detected in the G tumor and was greatly reduced in the normal prostate. Therefore, despite variable growth characteristics, there were few changes in membrane proteins between the solid tumors and between the tumors and normal prostate. Iodination of surface proteins of cultured cells from normal prostate and the G and H sublines also showed a high degree of homology. No consistent differences between cultured cell lines were noted.

Flow cytometric analysis of R3327 rat prostate adenocarcinoma grown in vivo and in vitro.

The Dunning R3327 transplantable prostate adenocarcinoma in the Copenhagen rat is an acceptable model for the human disease. The G-subline (a rapidly growing carcinoma) and the H-subline (a slow-growing, well-differentiated adenocarcinoma) represent the extremes of differentiation and growth rate of this tumor. Both sublines were found to have one population that was diploid and a second aneuploid population that was hyperdiploid in DNA content. The percentage of hyperdiploid cells was significantly higher in R3327-G tumors than in R3327-H tumors. The tumor cell population ratios were stable in vivo, but the in vitro culture conditions supported only cells with diploid DNA content following four to five subcultures. These predominantly diploid cultured cells, when injected into intact male rats, resulted in tumors that had both diploid and aneuploid cells.

Hormone sensitivity of the R3327-G rat prostate adenocarcinoma: growth rate, DNA content, and hormone receptors.

The growth of the R3327-G rat prostatic adenocarcinoma was significantly reduced when implanted in orchiectomized male rats (ORCH tumors). Tumors grown in intact animals (control tumors) had a doubling time of 7.4 days as compared to 9.2 days in ORCH tumors. A computer-based analysis of flow cytometric DNA histograms also detected significant differences between control and ORCH tumors. ORCH tumors were found to have 25% fewer cells with hyperdiploid DNA than control tumors (p less than 0.01). This androgen sensitivity in growth rate and the proportion of hyperdiploid cells were further reflected in the binding of [3H]methyltrienolone ([3H]-R1881) to cytoplasmic (cytosol) and nuclear tumor extracts. ORCH tumor cytosols had a [3H]R1881 binding capacity which was 70% lower than controls (6071 fmol/g tumor tissue). Nuclear [3H]R1881 binding in ORCH tumors was undetectable in seven of eight samples while in control tumors, binding was detectable in five of six preparations. Sucrose density gradient analysis showed that cytosolic [3H]R1881 receptors sedimented at 8.1 S in low salt and 4.6 to 3.3S in high salt. Nuclear [3H]R1881 receptors in high salt sedimented at 4.1 to 3.3S. Competition experiments using [3H]R1881 showed that [3H]-R1881 receptors were primarily androgenic, although some displacement by estradiol did occur. In contrast, [3H]estradiol binding was found to be highly specific. The binding capacity of [3H]estradiol in ORCH tumor cytosols was 30% higher than controls (962 fmol/g tumor issue), while binding to ORCH and control nuclear extracts was similar. These data suggest that the inhibition of androgen-sensitive R3327-G tumor cells was related to the concentration of androgen receptors and that this in turn was expressed as a reduction in the proportion of hyperdiploid cells.

Immunochemical studies of infectious mononucleosis. VIII. A glycoprotein from sheep erythrocytes with sialic acid-dependent receptor properties.

A glycoprotein was solubilized from sheep erythrocyte membranes with hot aqueous ethanol. The glycoprotein was purified by phosphocellulose chromatography, ethanol precipitation, lipid solvent extraction, and DEAE chromatography. In water solution the glycoprotein existed as globular aggregates with a diameter of 7.1 +/- 2.2 nm. In the presence of sodium dodecyl sulfate, 80% of the material exhibited a subunit m.w.app of 27,000. Approximately 10% of the material had a m.w.app of only 9000 and another 10% had a m.w.app of 35,000. All three fractions were reactive with Paul-Bunnell heterophile antibody from the sera of patients with infectious mononucleosis and with Limulus polyphemus lectin. These activities were destroyed by neuraminidase treatment. Complete inhibition of the rosetting of sheep red blood cells by 4 X 10(5) human peripheral blood lymphocytes was seen at 100 to 200 micrograms glycoprotein/ml. Neuraminidase-treated glycoprotein was not inhibitory. Pronase-derived sialoglycopeptide was inhibitory. Most likely, the receptor for lymphocytes resides in the carbohydrate portion of the glycoprotein. By using 125I-glycoprotein, binding studies were carried out that yielded an estimate of approximately 2 X 10(5) binding sites for sheep erythrocyte glycoprotein per lymphocyte. Purified glycoprotein contained 44.4% amino acid. Carbohydrate components and their molar ratios were sialic acid (1.0): galactose (1.0):N-acetylglucosamine (1.3): N-acetylgalactosamine (1.2).

Flow cytometric monitoring of R 3327 rat prostate carcinoma.

The R 3327 G tumor responds to estrogen in early stages, but relapses when estrogen therapy is continued beyond 50 days postimplantation. Measurement of DNA content per cell by flow cytometric analysis revealed two populations of cells in the tumors with ploidies of 2 c and 3.2 c. The proportion of aneuploid cells (3.2 c), determined from the flow cytometric DNA distributions, correlated well with tumor weight and age in control and estrogen treated animals. The simple parameter of per cent aneuploid cells thus adequately reflected the responsive and unresponsive states of tumors under hormonal therapy.

Flow cytometric analysis of the response of the R3327-G rat prostatic adenocarcinoma to endocrine manipulation.

The technique of flow cytometric DNA histogram analysis (FCM) shows there to be two distinct cell populations (diploid vs aneuploid) in the poorly differentiated R3327-G rat prostatic adenocarcinoma. The following study compares tumor weight measurements with several FCM computer-based methods designed to determine rapidly the proliferative status of tumors. Hypophysectomy, bilateral adrenalectomy, orchiectomy, sham operations, or diethylstilbestrol treatments were initiated when the tumors were palpable (day 21) and continued until the tumors were excised (day 52). Hypophysectomy, orchiectomy, adrenalectomy, and diethylstilbestrol treatments all resulted in significant inhibition by tumor weight. Quantitation of the percentage of mid-S phase aneuploid cells by summation gave the best correlation with tumor weight. Tumors grown in hypophysectomized, orchiectomized, adrenalectomized, or diethylstilbestrol-treated animals showed a significant reduction in the proportion of mid-S phase cells as compared with controls. The calculation of the percentage of all aneuploid cells was significantly reduced in hypophysectomy, orchiectomy, and diethylstilbestrol-treated animals. However, tumors grown in adrenalectomized animals were not significantly different from controls by this method. Adrenalectomy was found to be the least effective form of therapy, and this was reflected in all of the parameters measured. These data show that FCM analysis may be useful in the quantitation of prostatic carcinoma response to therapy.

Endocrine manipulation of the Dunning prostatic adenocarcinoma.

The Dunning animal model was used to evaluate endocrine management of prostatic adenocarcinoma. Hypophysectomy, alone or in combination, and orchiectomy plus stilbestrol were the most effective means of suppressing tumor growth. Medical adrenalectomy by aminoglutethimide administration was as effective as surgical ablation. Accessary organ weights bore no direct relationship to the inhibition of tumor growth.

Aqueous humor changes after experimental filtering surgery.

We studied aqueous humor of rhesus and owl monkeys for its effect on the growth of subconjunctival fibroblasts in tissue culture. Aqueous humor samples obtained before glaucoma surgery inhibited the initiation of growth of fibroblasts. However, postoperative aqueous humor samples supported growth of fibroblasts. The change in aqueous humor physiology lasted for up to two months after glaucoma surgery. Our study indicated that possibly material added to the postoperative aqueous humor inactivates an inhibitor normally present in primary aqueous humor. An alternative explanation would be that primary aqueous humor, in contrast to secondary aqueous humor, lacks sufficient nutrient material to support fibroblast growth in tissue culture.

The effect of aqueous humor on the growth of subconjunctival fibroblasts in tissue culture and its implications for glaucoma surgery.

We used aqueous humor from cataract patients and glaucoma patients as a medium for tissue culture of subconjunctival fibroblasts. The aqueous humor from cataract patients consistently inhibited the growth of their own subconjunctival fibroblasts, whereas that of some of the glaucoma patients did not. We found a significant correlation between the success of the filtering operation and the ability of that patient's aqueous humor to inhibit the growth of fibroblasts in tissue culture.

Establishment of primary cell cultures from normal and neoplastic human prostate gland tissue.

Explants of embryonic, normal adult, and tumor-containing prostate gland tissue in culture produced outgrowths of cells having an "epithelial" appearance. The fetal cells exhibited by far the highest potential for growth in vitro. Growth of prostate gland adult cells did not appear to depend on the age of the donor. Ultrastructural characteristics of cells from normal human prostates differed from cells derived from adenomatous prostate glands, which had many cytoplasmic characteristics similar to those described in solid tissues. These studies suggest that the characteristics of prostate gland cells in explant primary culture are similar to those in the parent tissues.