Improved method for processing autoradiographs of cells grown on multiwell plates.

A modification of an established procedure for autoradiographic processing of cultured cells is described. This method eliminates the need for pipetting each individual well and also for cutting and dismantling the multiwell plate for slide preparation. In this procedure the entire plate can be processed as a single unit and the cells can be analyzed in situ, thus eliminating the time consuming pipetting and cutting procedures. Furthermore, the entire experiment can be filed without use of additional slides or storage boxes. Hence, this is a simpler, time conserving, and economical way to process large numbers of cultures for thymidine labeling indices.

In vitro and in vivo responses of a murine transitional cell carcinoma to doxorubicin, mitoxantrone and aclacinomycin-A.

In vitro and in vivo effects of mitoxantrone, aclacinomycin-A and doxorubicin were examined in a transplantable murine transitional bladder carcinoma, FCB. The in vitro parameters used included monolayer growth kinetics, tumor stem-cell colony formation and autoradiographic analysis of thymidine labeling. Monolayer growth kinetics revealed that both mitoxantrone and aclacinomycin-A resulted in reductions in FCB cell growth, which were significantly higher (41% and 65%, respectively) than those seen with doxorubicin treatment (22%). Similarly, by the stem-cell assay, an increased reduction in colony formation was seen in aclacinomycin-A (98%) and mitoxantrone (91%) treated cultures when compared with doxorubicin (51%) treated cultures. Autoradiographic data revealed that 24-h exposure with both aclacinomycin-A and mitoxantrone significantly inhibited thymidine incorporation (98% and 80% respectively), which was an increase over doxorubicin (19%). In vivo studies revealed that aclacinomycin-A treatment increased the mean life span of C57BL mice by 60.6% when compared with a 33.6% increase in doxorubicin-treated animals and a 19.7% increase in mitoxantrone-treated animals. Both the in vitro and in vivo data suggest that aclacinomycin-A is a superior drug when used against this specific murine bladder tumor cell and that further testing of this agent for its efficacy in other urologic tumors is justified.

Determination of [3H]TdR-labelling indices of cultured cells grown on detachable chamber slides.

The in vitro effects of xenobiotic agents on the incorporation of DNA precursors was determined using a detachable chamber slide assembly. The technique presented allows for the comparative assessment of several agents, even when a limited number of cells is available. The use of a glass slide also potentiates the use of higher magnifications with a greater resultant resolution than that achievable with past methods.

A convenient method for in situ processing of cultured cells for cytochemical localization by electron microscopy.

The effects of various exogenous agents on subcellular structures is of importance to many investigators and can be critically evaluated by the use of cytochemical techniques and transmission electron microscopy. Therefore various cell culture techniques have become increasingly important in biological research in order to help determine these effects. Presently, most existing methods for processing anchorage-dependent cultures for electron microscopy utilize cells grown on either glass or plastic substrates. Therefore various coating substances have been applied to the culture surfaces to facilitate removal of the sample, however, incomplete separation and sample fracture often results. Here we present a simple method of in situ processing of samples for electron microscopy involving the use of detachable chamber slides. This method allows for the use of a quick processing procedure that results in a complete separation of the sample from the glass slide.

Growth and ultrastructural responses of T-47D human breast tumor cells to treatment with mitoxantrone.

Mitoxantrone is a new anthracenedione derivative that suppresses cell proliferation in the T-47D human breast tumor cell line as revealed by colony-forming assay in soft agar and growth study in monolayer culture. One-hour drug exposure at 10(-9) M, 10(-7) M, and 10(-5) M reduced colony formation to 30%, 3%, and 0.5% of the control value, respectively. Little inhibition of cell growth was observed in monolayer cultures after 24 hr treatment with 10(-9) or 10(-8) M mitoxantrone, but a sharp decline occurred between 10(-6) M and 10(-5) M. Cytotoxicity was evident after 24 hr treatment with 10(-4) M drug; fewer than 10% of the cells survived. [3H]thymidine incorporation declined rapidly between 10(-9) M and 10(-6) M, revealing the potent inhibitory effect of mitoxantrone on DNA synthesis and cell proliferation. Ultrastructural examination revealed nucleolar alterations including dissociation and segregation of fibrillar and granular components, suggesting that this organelle is a principal intracellular target of mitoxantrone.

Response of 7,12-dimethylbenz(a)anthracene-induced rat mammary tumor cells to tamoxifen, in vitro.

Primary cell cultures from a density-defined cell subpopulation of the DMBA-induced rat mammary tumors were exposed to tamoxifen during their log phase of growth. Growth inhibition and the ultrastructure of surviving cells were examined along with the influence of this antiestrogen on the secreted proteins as determined by pulse labeling with [35S]methionine and fluorography. Cell growth was remarkably inhibited at clinically achievable concentrations. However, ultrastructural changes in the surviving cells were minimal, the most noteworthy being the accumulation of myelin bodies. Protein secretion was affected in the defined subpopulation of several tumors by the reduced production of a high-molecular-weight protein. These tumors may represent a population of estrogen-sensitive tumors within the DMBA-induced mammary tumor model.

Sequential study on the influence of adriamycin on cell proliferation and ultrastructure of cultured mammary tumor cells.

The time-dependent cytocidal and growth inhibitory effects of Adriamycin (ADM) on monolayer cultures of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumor cells were analyzed. The inhibitory effect on cell proliferation was assessed by colony formation in soft agar. Growth inhibition and [3H]thymidine labeling indices clearly demonstrate a dose-dependent antimitotic and cytotoxic effect of the drug. At low concentrations (10(-9)-10(-8) M), 90-100% of cells survived 24-hr exposure. At a higher concentration (10(-5) M), 75-80% of cells survived after 8-hr exposure; by 72 hr only 20-30% of the cells remained. Autoradiographic examination of the pulse-labeled cultures demonstrated no change in the proportion of cells in S-phase during the first 4 hr of treatment. Subsequently DNA synthesis was completely abolished and remained inhibited for the duration of the experiment (72 hr). Clonogenic assay revealed a complete arrest of growth in cells exposed to 10(-5) M ADM and greater than 60% inhibition of cell proliferation at 10(-7) M. Ultrastructural changes were not observed in cells during the first 4 hr of treatment; however, after 8 hr most surviving cells exhibited alterations in nuclear chromatin. The surviving cells showed mitochondrial degeneration, myelin body formation, and vacuolization of the endoplasmic reticulum. This study shows the potential usefulness of the primary culture system in drug evaluation. In addition, serial observation of the effects of ADM revealed a cell subpopulation of the primary culture with differential sensitivity to the drug.

Pharmacological therapy for proliferative vitreoretinopathy.

We developed an animal model in the rabbit eye with intravitreally injected heterotransplanted bovine retinal pigment epithelial cells to test a pharmacological agent which would reduce extracellular matrix formation. Results with 20 and 60 micrograms of cis-hydroxyproline showed a 48% decrease in the rate of traction retinal detachments.

Verapamil enhanced in vitro chemosensitivity of a murine bladder carcinoma, FCB.

The in vitro enhancement of chemotherapeutic efficacy by verapamil, a calcium antagonist, was assessed using FCB, a transplantable murine transitional cell carcinoma. Exponentially growing FCB cells were partially resistant to treatment with both thiotepa (10(-4) M) and Adriamycin (10(-5) M), however, there was a significant reduction in cell growth when either agent was administered in combination with verapamil (10(-5) M); the effect was evident over a wide range of drug concentrations (10(-4) - 10(-9) M). There was also a pronounced inhibition of DNA precursor incorporation when verapamil was used in combination with either agent. Fluorometric analysis of Adriamycin uptake indicated that verapamil caused an increase in the intracellular concentration of the agent. The data presented are consistent with the postulate that verapamil enhances chemotherapeutic efficacy by altering cellular permeability to the cytotoxic agents. Our study indicates that the use of verapamil in combination with cytotoxic agents for intravesical chemotherapy of bladder tumors may prove to be beneficial in human patients.

In vitro chemosensitivity of J-82 human bladder cancer cells.

While chemotherapy offers a valuable adjunct to surgery in the management of intravesical bladder cancer, an accurate in vitro predictive test for chemosensitivity has yet to be developed. Drug sensitivity of the human bladder cancer cell line J-82 was assessed using monolayer, stem cell and [3H]thymidine incorporation assays. The 72-h monolayer assay provided a rapid reflection of in vitro drug sensitivity and when combined with the labeling index the results generally paralleled those obtained with the soft agar stem cell assay without the associated large commitment of time and labor. It is suggested that 72-h monolayer assay alone or in combination with [3H]thymidine labeling index may offer valuable insight into the chemotherapeutic response of bladder tumors.