Jerome J. Freed

Ameboid movement induced in cultured macrophages by colchicine or vinblastine

Abstract Mouse peritoneal macrophages cultured for 48 h, when subjected to colchicine or vinblastine treatment and examined by time-lapse microscopy, reveal a shift in cellular locomotion. The movement of the cells is altered from a gliding form of locomotion to an induced ameboid (flowing) form. Electron microscopy shows that cytoplasmic microtubules disappear from the drug-treated cells and the normal polarization of cytoplasmic organelles is disrupted; the ameboid cells also contain numerous microfilaments of both the 55 AU subcortical type and the 100 AU colchicine-induced type. The interpretation offered for these observations is that contractility of the cytoplasmic cortex is normally opposed by the interphase microtubule array; in the absence of this cytoskeletal structure differences in cortical tension may lead to cytoplasmic streaming and consequent ameboid movement.

Chromosome aberrations in cultured cells deprived of single essential amino acids

Abstract When cultured Chinese hamster cells (pseudodiploid line C14FAF28) were exposed to growth medium deficient for any single essential amino acid, the cell cycle was inhibited during S-phase; incorporation of 3H-thymidine into nuclear DNA declined markedly. After restoration of an adequate concentration of the omitted amino acid, the division cycle was completed. The metaphases of this division contained a high frequency of chromosome abnormalities, including chromosome breaks and exchanges, a major class of chromatid breaks and exchanges, and endoreduplication. A high frequency of breakage and exchange was associated with severe fragmentation, superficially resembling virus-induced pulverization, but distinguishable from the virus-induced phenomenon by non-labelling after terminal exposure to 3H-thymidine. The yield of aberrations increased progressively as a function of the time during which the cells were held in the inhibited state. When cells were cultured at a growth-limiting concentration of arginine, aberration-containing metaphases were revealed after addition of supplementary arginine. It is proposed that interruption of DNA replication, and consequent chromosome aberration, are the result of the inhibition of protein synthesis. Chromosome aberrations arising in this way may play a role in apparently spontaneous karyotypic remodeling in serially propagated cultured cell lines and progressing neoplasms. Cycling of cultured cells through amino acid deprivation is proposed as a technique for deliberately increasing the rate of chromosome alteration in genetic experiments.

Altered movement of endosomes in colchicine-treated cultured macrophages☆

The effect of colchicine on the movement of phagocytic vacuoles (endosomes) has been studied by time-lapse cinemicrography and electron microscopy of cultured mouse peritoneal macrophages. In normal macrophages, ingested heat-killed E. coli are transported by independent saltatory displacements from the cell periphery to the cytocenter; in cells treated with colchicine so as to contain few intact microtubules these saltatory movements do not occur. Bacilli are incorporated and endosomes are formed essentially as in untreated cells, but endosome movement is passive. Endosome movement in colchicine-treated cells apparently results only from the flow of cytoplasm and ameboid movement induced by the drug. Changes in the distribution of acid phosphatase during phagocytosis (degranulation, appearance of diffuse enzyme staining and acid phosphatase staining of ingested bacilli) appear similar in normal and microtubule-deprived cells. Thus, microtubule-associated functions do not appear to be essential for formation of endosomes or for addition of lysosomal acid phosphatase; saltatory movements are not an obligate part of phagocytosis.

Remnant motility of macrophages treated with cytochalasin B in the presence of colchicine

Abstract We have previously observed that mouse peritoneal macrophages cultured for 48 h and treated with colchicine to depolymerize cytoplasmic microtubules become ameboid and cease to migrate by gliding on the substratum. We have now found that when such cells were further exposed to both colchicine and cytochalasin B, the induced ameboid movements were reversibly inhibited. Cells treated concomitantly with both drugs did not become motionless, but exhibited a remnant motility that took the form of zeiosis (blebbing). The zeiotic blebs contained ribosomes and fibrous material, but lacked organized microfilament arrays and rarely included other cytoplasmic organelles. Zeiosis appears to be a form of surface movement independent both of cytoplasmic microtubules and of the cytochalasin-sensitive contractile system. These observations imply an additional mechanism that can reversibly alter the form of the cell.

Characteristics of Cell Lines from Haploid and Diploid Anuran Embryos

Haploid anuran embryos can readily be obtained by the classical methods of experimental embryology and provide a source of tissue for the initiation of haploid cell cultures. In order to exploit the obvious advantages for genetic experimentation of cell populations with a single chromosome set, we have for some time been studying cell lines initiated from haploid as well as diploid frog embryos (1). Since our initial publication in 1962, we have evaluated a number of growth media; the most useful appears to be that of Leibovitz (2) as modified for amphibian cells by Balls and Ruben (3), which we have employed since 1966. Using this medium, 3 5 lines have been initiated by passaging primary cultures. Of these, 23 have successfully been maintained in serial culture, i.e., carried through 10 or more passages. Clonal isolates have been obtained from some of these lines and passaged separately. Thus, 3 6 distinct lines are at present maintained in frozen storage or continuous culture. Our purpose in this paper is to describe how such lines are initiated, the morphological changes which the cells undergo, their chromosome constitution, and their properties as experimental material. We will consider in detail features of four lines of particular interest because of their origin or behavior.

Chapter 2 Culture Methods for Anuran Cells1

Publisher Summary This chapter describes the procedures used for initiating and maintaining anuran embryo cell lines and clones. The material is presented in detail to serve as a useful guide in the laboratory. The specific methods described in this chapter have been developed to exploit the unique advantages that frog cells offer for genetic studies. Haploid frog embryos, produced by methods derived from experimental embryology, yield adequate amounts of tissue for the initiation of cell lines. Cells with a single chromosome set are particularly useful for investigating somatic gene changes and for the isolation of genetic variants. As cultures may be maintained at room temperature, they are well suited for direct observation of mitosis; the cells and chromosomes are large enough for interesting observations with the high-dry objective. The Briggs and King nuclear transfer technique provides a means of testing genetic variants in the developing embryo. The relatively small number of large chromosomes facilitates cytogenetic work.

Double‐Beam Vibrating Mirror Flying Spot Scanning‐Integrating Microspectrophotometer

Serial measurements of integrated ultraviolet absorbance of individual living cultured animal cells are possible with a double‐beam scanning microscope designed to maximize the signal‐to‐noise ratio obtained for a given radiation flux traversing the sensitive specimen. A 192‐line raster (2 sec/frame), scanned in the specimen plane by a focused spot of monochromatized radiation, is reconstructed by video technique to provide an image display and permit manual delineation of photometric fields of arbitrary shape, corresponding to the shape of the specimen. Analog circuits produce a read‐out proportional to the integrated absorbance present in the photometric field, unaffected by inhomogeneity in the distribution of absorbing material.

FROG RENAL ADENOCARCINOMA: CYTOLOGICAL STUDIES IN SITU AND IN VITRO*

Introduction The Luck6 tumor, the common renal adenocarcinoma of the grass f rog R a m pipiens, has long been believed to have a viral etiology. Cell f ree materials prepared from the tumors increase the rate of appearance of new adenocarcinomas when injected into adult frogs (Luck6, 1952 ; Duryee, 1956; see review by Rafferty, 1964). In some tumors, intranuclear inclusion bodies occur (Luck6, 1952), and examination of such tumors with electron microscopy reveals abundant virus particles of the herpesvirus-type (Fawcett, 1956 ; Zambernard & Mizell, 1965 ; Lunger e t al., 1964). However, in other tumors having the same histological structure, in which inclusion bodies a re lacking, no virus is demonstrable by electron microscopy. It has been pointed out t ha t tumors of the latter type a re more likely to contain mitotic figures (Fawcett, 1956; Rafferty, 1964). Two types of Luck6 tumors may thus be distinguished: a “viral” type and a “mitotic” type. Rafferty (1964) has recently proposed a hypothetical life cycle to account for these differences. He suggests t ha t during the summer the tumor proliferates mitotically ; when the animal becomes inactive during the cold winter months, viral inclusions appear. As the f rog resumes activity in the warmer months, cells containing inclusion bodies disappear, and the tumor resumes proliferative growth and finally becomes large enough to result in the death of the animal. Evidence in support of this view comes from the fact tha t “viral” tumors are found in frogs maintained at refrigerator temperatures or collected as they emerge from hibernation; while frogs collected in the field or maintained at laboratory temperatures have tumors of the “mitotic” type. I n experiments reported elsewhere in this monograph, Rafferty (1965) describes the conversion of “mitotic” tumors, as determined by biopsy, to “viral” tumors a f te r prolonged storage of the host animals in the cold. If the life cycle proposed by Rafferty is adopted as a working hypothesis, some fur ther questions immediately arise. First , how is viral replication suppressed during the summer? Is virus associated in any way with the mitotic summer tumors ? Are the cells containing viral inclusions converted to the mitotically proliferating type, or a re they replaced by a new cell population ?

Frog Embryos (Haploid Lines)

Publisher Summary This chapter covers the methods for culturing of frog embryos. These methods can be used to initiate lines of either haploid or diploid amphibian cells. The maximum rate of cell multiplication occurs between 25°C and 28°C, with lethal effects at higher temperatures; cultures are kept in low temperature incubators. The most favorable pH is 7.5. Multiplication is more rapid at pH 7.0, but the cells are more prone to lysis. Large frogs suitable for breeding are obtained from the commercial suppliers and maintained in the laboratory in tap water at 5°C. Gravid females captured in the fall are the best source of healthy embryos. Ovulation is hormonally induced by injecting a single frog pituitary gland intraperitoneally through a No. 23 hypodermic needle. Androgenetic haploids are obtained by the technique of Porter. Gynogenetic haploids may be obtained in large numbers by photodynamic inactivation of sperm. The quality of the eggs appears to be a factor in the success of the culture procedures and can be determined in advance by the frequency of normal cleavage of diploid embryos.

28th Annual Meeting Tissue Culture Association, inc.

Cell biology at the present time is entering upon the reductionist phase, the stage in the evolution of a scientific discipline when it passes from the description of phenomena and an analysis of their interrelations to the investigation of the fundamental mechanisms at work. Genetic, biochemical and molecular biological investigations of the cells and tissues of higher organisms have been greatly facilitated by the coming of age of methods for in vitro cell and organ culture. In such studies, culture systems can now be made to yield information heretofore only available from microorganisms; and the presentations in this symposium have been chosen to reflect the current state of this molecular approach to the problem of cellular control mechanisms. The first aspect of cellular control mechanisms discussed here is the control of cellular proliferation. In ceils of higher organisms, proliferation is determined not only by the availability of nutrients, but also by the cells capacity to respond to specific signals. Such signals (e.g. hormones and growth factors} are presented by the cellular environment and can be studied effectively in the controlled milieu of a culture system. The state of this approach is discussed in the first three papers of this series. A salient feature of cellular proliferation is the replication of DNA; this process as it occurs in mammalian cells has been dissected through use of imposed cell cycle synchrony, as described in the fourth paper. The second topic considered here is cellular differentiation, a hallmark of which is the synthesis of those specialized gene products that mark the assumption of specialized cell functions. Here, too, the use of culture methods now permits an attack on the molecular mechanisms that control these altered patterns of gene expression; the remaining papers report on this approach. The response of an organ culture system to hormonal stimulation in vitro is the basis of an analysis of the altered transcription that leads to the secretion of milk proteins. Use of mutant ceils derived from cultured cell lines permits a dissection of the relation of cyclic AMP to hormonal response. Finally, we have two contributions dealing with erythroleukemic cell cultures, a model system of differentiation to which has been applied a variety of sophisticated research tools.