Arline D. Deitch

A stable propidium iodide staining procedure for flow cytometry.

A propidium iodide (PI) staining procedure is described in which 50 micrograms/ml PI in 10(-2) M Tris, pH 7.0, with 5 mM MgCl2 is used to stain murine erythroleukemia cells (MELC) grown in suspension culture as well as single cell suspensions derived from rat kidney adenocarcinoma and human prostatic carcinoma. Specificity of staining of nuclear DNA is achieved by enzymatic removal of RNA using RNAse in the staining solution. Virtually identical histograms, with the same G1 peak height and closely similar coefficients of variation (CVs), are obtained using a wide range of RNAse concentrations on replicate samples of MELC if the incubation times are sufficiently prolonged when employing the lower enzyme concentrations. For 1 mg/ml RNAse on logarithmically growing MELC, 30 min incubation at 37 degrees C is needed to obtain a maximum G1 peak height and optimal CV and there is no significant change in the histogram if the incubation is prolonged to 4 hr. For every 4-fold decrease in RNAse concentration, the incubation time at 37 degrees C must be doubled to obtain the same maximal G1 peak height and optimal CV. Unfixed cell preparations, whether derived from suspension or monolayer cultures or from solid tumors, are stable for 2 or more weeks if stored at 4 degrees C between flow cytometric analyses and histograms are usually only minimally altered if the stained cell samples are stored for 1-2 months at 4 degrees C. Sample decay is associated with bacterial contamination. If sterile preparative techniques are used initially, subsequent contamination of the stained preparations may be minimized by adding sodium azide to the stained samples at 0.1% without influencing fluorescence intensity. Glycerine may be added to 10% and the samples slowly frozen for storage without altering DNA histogram shapes. The simplicity of sample preparation and the stability of the resulting stained cell samples makes this procedure suitable for repetitive comparative sampling of tissue and cell populations over prolonged time spans.

Flow cytometry: Role in monitoring transitional cell carcinoma of bladder

The ability to detect and monitor the course of transitional cell carcinoma (TCC) of the bladder using DNA histograms obtained from flow cytometry was studied. Voided urine and barbotage specimens were collected from patients with active TCC or a past history of TCC. These specimens were submitted to routine cytologic and flow cytometric analyses. Samples were considered to be positive if they met one or more of three criteria: if they had aneuploid or tetraploid peaks, if the DNA index of the major G1 peak was shifted more than 10 per cent from that of diploid cells, or if 15 per cent or more of the cells fell to the right of the major diploid G1 cell population thereby constituting a significant hyperdiploid cell population. Using these methods for patients with active disease, the detection rate was 91 per cent. In patients with a past history of TCC, positive histograms preceded the appearance of visible tumor in one third of the cases. Flow cytometry proved to be an excellent way of following patients with a past history of TCC or of screening patients suspected of having active disease. Following this protocol, few biologically active tumors go undetected. However, in 112 patients without a history of bladder cancer, the false positive or suspicious rate was 38 per cent. Before flow cytometry can be recommended as a widespread screening method for patients thought to be at risk of TCC of the bladder developing, this suspicious group will have to be eliminated.

Cell volume changes in relation to the cell cycle of differentiating erythroleukemic cells

Abstract Murine erythroleukemic cells (MELC) were synchronized by sequential exposure to thymidine and hydroxyurea. Upon removal from hydroxyurea, cells cultured with or without agents that induce erythroid differentiation, such as hexamethylene bisacetamide (HMBA) or dimethylsulfoxide (Me 2 SO), proceed through S, G2 and mitosis with the same kinetics: S phase averages 5 h and G2 plus mitosis, 2 h. Cells cultured with HMBA and Me 2 SO remain in the subsequent G1 for 5–7 h, compared with an average of only 3 h for cells cultured without inducer. Modal cell volume doubles as the cells proceed from G1 to G2. During the inducer-mediated prolonged G1, MELC retain a small cell volume. In cultures of non-synchronous MELC, inducers also increase the G1 fraction, as well as the proportion of small cells. An Me 2 SO-resistant MELC variant (DR10), cultured with Me 2 SO, shows little prolongation of G1 and little difference in the modal cell volume compared with cells without inducer. However, HMBA, which induces differentiation of DR10 cells, prolongs G1 and increases the proportion of small cells. These studies indicate that early changes in cell volume associated with induction of MELC to differentiate, in large part reflect alterations in the cell cycle. Evidence is presented which suggests that only one round of DNA synthesis in the presence of inducer may be necessary to initiate differentiation.

Effects of cordycepin on microtubules of cultured mammalian cells.

Abstract Metaphase cells accumulate in HeLa and Vero cultures exposed in G2 to cordycepin (3′deoxyadenosine, 3′dA) at doses of 25–100 μg/ml. The arrested cells have small spindle zones and reduced numbers of interpolar or non-chromosomal spindle microtubules. While these concentrations of 3′dA have been used to suppress mRNA synthesis, the mitotic arrest does not appear to result from inhibition of RNA synthesis. Synchronized Vero cells treated in G2 with sufficient concentrations of actinomycin D to suppress transcription of virtually all RNA do not subsequently become arrested in mitosis. It is proposed that cordycepin interferes with the formation or normal functioning of the mitotic spindle. The microtubules of interphase cells may also be affected by cordycepin. The amount of microtubular crystals formed after exposure of several different cell types to vinblastine sulfate is markedly reduced after pretreatment with cordycepin.

Enhancement of viral infectivity by cytochalasin

Abstract Cytochalasins B and D enhance the infectivity of poliovirus; a 5-fold increase in viral titer occurs on treatment of cells with 4–6 μg/ml of either congener. At lower concentrations, however, CD is more effective than CB in increasing the infectibility of cells. The enhancement of viral infectivity occurs whether CD is applied to the cells before and during viral attachment, or after penetration has occurred. CD does not affect the attachment, penetration, or elution of radiolabeled poliovirus, and only minimally stimulates uncoating. Enhancement of infectivity progressively declines as the time of addition of cytochalasin after viral penetration is prolonged. We conclude that cytochalasin affects some event(s) early in the eclipse period of polioviral development. Pretreatment of Vero cells with dibutyryl cyclic adenosine 3′:5′-monophosphate antagonizes the effect of CD on the infectivity of poliovirus. Cytochalasin also increased the infectivity of other RNA viruses including parainfluenza virus. However, no augmentation of infectivity is found with vaccinia, and infectibility with adenovirus is decreased by CD.

Modification of Plastic Culture Dishes for High-Resolution Microscopy of Living Cells

The plastic Cooper-type of dish has been adapted to high power, high resolution phase-contrast microscopy by drilling a 3/4 inch hole in the center of the bottom of the dish and cementing a 30 × 30 mm coverslip over this hole. Once the trypsinized cell suspension has been added and the dish closed, the top of the dish is sealed to the bottom by means of a roll of plasticene to prevent loss of CO2 and water. The preparation is maintained in a stage incubator. The same field can be observed with an inverted phase contrast microscope for 3 to 4 days. Either high dry (40-45x) or oil immersion objectives may be used to study the cells. The high resolution obtainable is especially useful in evaluating the effects of viruses or chemical agents on living cells in monolayer cultures.

The influence of cytoreductive surgery on the response to chemotherapy of a rat renal cancer

SummaryThe potential ability of cytoreductive surgery to increase the effectiveness of chemotherapy (vindesine) was tested utilizing male Wistar Lewis rats transplanted simulataneously with intraperitoneal and flank implants of a spontaneously arising renal adenocarcinoma. Cytoreduction was accomplished in some animals by removing the flank tumor 5–7 weeks following implantation; all animals received vindesine (IP injection of 0.5 mg/kg on two successive weeks). While vindesine reduced tumor growth, in no case did the addition of cytoreductive surgery enhance the effect of chemotherapy. The addition of cytoreductive surgery to marginally effective chemotherapy was found to be ineffective or even detrimental.

Cytochalasin D enhances infectivity of poliovirus.

Summary Treatment of permissive or semi-restrictive cell lines with cytochalasin D (CD) enhances their susceptibility to infection with poliovirus. A two-to-threefold increase in the number of productively infected cells, as well as an augmented viral yield and a more rapid onset of cytopathic effects occur in drug-treated cultures. CD exerts its potentiating effect early after adsorption either during or, more probably, after viral penetration. This work was supported by grant VC-76 from the American Cancer Society and grants CA-13835 and AI-06801-07 from the National Institutes of Health. We are grateful to Stanley G. Sawicki for many fruitful discussions and helpful suggestions.

Alterations of physical and biochemical parameters of the R3327-CP rat prostate adenocarcinoma following hormonal manipulation of the host.

Several physical and biochemical parameters of a rapidly growing, hormonally responsive, poorly differentiated strain of Dunning R3327 rat prostatic adenocarcinoma (the CP strain) were monitored for 1 month during growth in control and hormonally manipulated male Fischer X Copenhagen rats. The tumor was implanted into control rats and into rats 1 month following orchiectomy. Twenty-nine days following tumor implantation, 1 group of unoperated rats was orchiectomized while the rats implanted subsequent to orchiectomy were repleted with pharmacological doses of testosterone. At 2 and 4 weeks following treatment, half the original number of rats from each group were sacrificed and the growth rate (doubling time), per cent of aneuploid cells and androgen receptor levels (total, cytoplasmic and nuclear) were determined for each tumor. Orchiectomy increased tumor doubling time, while testosterone repletion decreased it, demonstrating the hormonal dependence of this tumor strain. Orchiectomy also decreased the levels of aneuploid cells in the tumor; however, repletion of testosterone to rats orchiectomized prior to implantation did not restore the aneuploid cell number to control levels. A sensitive indicator of the hormonal status of the tumor was the per cent of androgen receptors in the nucleus. Tumors grown in rats orchiectomized after implantation had the lowest percentage of androgen receptor in the nucleus while orchiectomized rats repleted with testosterone had the highest percentage. Comparison of the levels of androgen receptors in the tumors from the various groups (androgen receptor per gram of tissue) unexpectedly revealed that tumors grown in the orchiectomized rats had slightly higher total receptor levels than did control tumors, while the tumors of orchiectomized rats repleted with testosterone had lower amounts than did the control tumors. In contrast to these findings, the prostates of orchiectomized rats replenished with testosterone had higher levels of total androgen receptor than did the prostates of control rats.

Physikalisch-optische Methoden in der Histochemie: Vergleich der Ergebnisse mit verschiedenen unabhängigen Methoden

Die physikalisch-optischen Methoden umfassen heute schon einen grosen Teil des elektromagnetischen Spektrums: Rontgenhistoradiographie, Photometrie im ultravioletten und sichtbarem Licht, Interferenzmikroskopie, Infrarotphotometrie. Die quantitativen oder halbquantitativen Substanzbestimmungen erfolgen mittels photometrischer Methoden, wobei die Lichtabsorption mit Hilfe des Mikroskopes gemessen wird. Verschiedene Fehlerquellen konnen zur Verfalschung der Mesergebnisse fuhren: 1. Meβ- und Apparatefehler, wie z. B. Schwarzschild-Villiger-Effekt, Grose der Mesblende, Hohe der Extinktionen u. a. 2. Objektbedingte Fehler: wie z. B. Streulicht durch Brechungsindexdifferenzen, ITherlagerungen von Absorptionen verschiedener Substanzen u. a. 3. Farbungsfehler: wie z. B. ungenugende Stochiometrie, Artefakte verschiedenster Art. — Diese kurze Aufzahlung der Fehlerquellen zeigt, das die Mesergebnisse mit anderen unabhangigen Methoden uberpruft werden mussen.