James S. Murphy

Interferon effects on the growth and division of human fibroblasts.

Abstract The overall rate of proliferation of human fibroblasts in culture is reduced at interferon concentrations greater than 40 international reference units (U)/ml. Inhibition is near maximal at 640 U/ml, at which concentration the doubling time between 24 and 72 h after beginning of treatment is increased 2–3 times over the control value. Inhibition of cell proliferation was not readily reversible upon removal of interferon and refeeding of cultures. Study of the mitotic behavior of individual cells showed that the first intermitotic interval after beginning of treatment with interferon (640 U/ml) was prolonged in about two-thirds of the cells. In this fraction, many cells failed to divide again after the second post-treatment mitosis, while others exhibited a progressively increasing intermitotic interval with subsequent divisions. One-third of the interferon-treated fibroblasts initially divided at a rate similar to the rate of proliferation of control cells, but subsequently these cells also slowed down and finally stopped dividing. After treatment at 640 U/ml for 3 days, the rates of DNA, RNA, and protein synthesis were depressed to 86, 75, and 64% of control values, respectively. However, the interferon-treated fibroblasts had grown larger than control cells as indicated by the following parameters: cell attachment area, 165%; volume, 131%; DNA content, 130% and protein content, 150%. Thus, interferon does not prevent cell growth, but interferes with cell division.

A phage-associated enzyme of Bacillus megaterium which destroys the bacterial cell wall

Abstract A virulent Bacillus megaterium phage G, which produces a spreading halo around its plaque, has been isolated. The halo is caused by a lytic substance which diffuses faster than the phage. Broth lysates of phage G also contain a lytic substance which has the characteristics of an enzyme and which dissolves B. megaterium cell walls. The enzyme has been characterized, separated from phage, and purified to the extent of increasing the specific activity 118 times. It is rapidly destroyed at 65°;, and has a temperature coefficient of activity of 1.54 and a pH optimum of about 4.5. In pH 8.6 buffer, it migrates electrophoretically to the anode in a single peak. It is resistant to trypsin, papain, deoxyribonuclease (DNAse), and ribonuclease (RNAse) but is destroyed by chymotrypsin. The action of the enzyme on cell walls is accompanied by the appearance of nondialyzable reducing sugar. The enzyme appears not to be serologically related to phage. Data are presented to show that the enzyme may be found in the cells in increasing amounts up to the time of lysis, probably starting shortly before the first mature phage particles are formed; that the rate of premature lysis brought on by the addition of cyanide correlates with the amount of enzyme present at any time; that proflavine, at a concentration which partially inhibits phage growth, allows development of the full titer of the enzyme; and that, by 3 seconds after the onset of phage lysis, the cell wall has been destroyed. No enzyme activity seems to be present in purified phage preparations at concentrations up to 3 × 1013 per milliliter. It is hypothesized from the above data that the enzyme is produced in the cell under the influence of phage and, in this case, may function in an escape mechanism for the phage by destroying the cell wall.

Dissociation between early loss of actin fibres and subsequent cell death in serum-deprived quiescent Balb/c-3T3 cells

Serum withdrawal from either growing or quiescent Balb/c-3T3 murine fibroblasts causes a loss of F-actin fibres and focal adhesions within 30 min. Cells that are growing survive serum deprivation, whereas the great majority of density-arrested quiescent cells die during a period of up to 5 h from serum withdrawal. During this time an approximately constant fraction of the quiescent cell population dies per unit time. The population half-life is 60-70 min during this time. Addition of an appropriate cell growth factor or second messenger agonist at the time of serum withdrawal or within 2 h after serum withdrawal protects a similar fraction of viable cells. These findings suggest a model according to which withdrawal of serum (i.e. growth factors) initiates the death process in cells of the population with kinetics that approximate first-order kinetics. We postulate that appropriate growth factors or second messenger agonists block the initiating event that starts the cell death process.

Cytokines in breast cancer cell dyshesion

While the evidence for the genetic basis of cancer is mounting [reviewed in 1-3], at the same time it is becoming apparent that growth factors and cytokines may play critical roles in determining the phenotypic expression of cancer [4-6]. Interleukin-6 (IL-6) decreases intercellular and substratum adhesiveness in lines of human ductal breast carcinoma cells [7-12]. Dyshesiveness is a cardinal pathological feature of breast carcinoma [13]. Ductal breast carcinoma cells are morphologically heterogeneous not only within the same tumor, but also in sublines originating from the same line of tumor cells. In the Ro subline of the ZR-75-1 cells EL-6 converts cuboidal/polygonal cells to stellate cells with long processes [7]. The morphologically altered ZR-75-1-Ro cells separate from each other and display enhanced directional motility although their proliferation is suppressed. No evidence was obtained of growth factor-inducible cell-cell separation with transforming growth factor-α (TGF-α), TGF-β1, epidermal growth factor (EGF), or insulin-like growth factor (IGF-1) [9]. Colonies of ZR-75-1-Ro cells formed in the presence of acidic fibro-blast growth factor (aFGF) or basic fibroblast growth factor (bFGF) (10 or 100 ng/ml) did however show some evidence of cell-cell separation.

[48] Assay of the inhibitory activities of human interferons on the proliferation and locomotion of fibroblasts

Publisher Summary This chapter describes two approaches to the investigation of the antiproliferative action of interferon on human fibroblasts, which are determination of proliferation curves by cell counts or enumeration of mitoses and measurement of intermitotic intervals. Determination of the intermitotic intervals for individual cells by time-lapse cinemicrography reveals and makes possible a quantitative definition of the heterogeneous response of cells in a population to the antiproliferative effect of interferon. The chapter also describes a procedure for the measurement of the inhibitory action of interferon on cell motility, a procedure that can be applied to time-lapse cinemicrographs used for the study of the antiproliferative action of interferon. The technique described for serial cell counts permits convenient study of multiple cell types and of multiple interferon preparations. Relatively few cells are required for the determination of proliferation rates and of dose-response relationships. The counting technique is nondestructive; this means that the same cultures can be examined repeatedly at intervals of time, thereby avoiding the variation that arises when different sets of culture vessels need to be used for consecutive determinations.

The death rate of lysogenic bacteria after ultraviolet induction follows gompertz's law

Abstract After ultraviolet induction of Bacillus megaterium 899a, both the rate of bacteriophage release and the rate of bacterial lysis increase with kinetics similar to those found in autocatalytic reactions. The virus-release curve may be measured over a span of five log units. Evidence is presented that this phenomenon is not mediated by information exchange among bacteria and, therefore, is not dictated by an extracellular autocatalytic reaction. The results show that the kinetics of death in induced cultures are similar to the kinetics of death found in higher organisms where the death rate increases exponentially with the age of the population (Gompertzs law). In other words, when a culture of lysogenic bacteria is induced, it acquires a defined lifespan and proceeds to die with kinetics obeying Gompertzs law. A possible mechanism is discussed in an attempt to explain the results at the molecular level.