Normal Animal Cells Research Articles

Biomedical gerontology. Current theories of biological aging.

Several lines of evidence have led to the notion that biological aging occurs as a result of changes in information-containing molecules either at the genetic or epigenetic level. The error theory, the redundant message theory, the codon restriction theory, and the transcriptional event theory represent the major current conceptualizations of biological aging as held by most gerontologists. The finding that cultured normal human and animal cells undergo a finite number of population doublings in vitro has provided new insights into age changes at the cellular level. The number of mitotic events that cultured normal animal cells can undergo appears to be inversely related to the age of the donor. A direct proportionality exists, however, between the mean maximum life-span of a species and the number of population doublings that their cultured embryonic cells will undergo. The several biochemical decrements known to occur prior to the cessation of mitotic activity in vitro are thought to herald those manifestations of senescence seen in the whole animal. Yet to be explained is how those cell classes such as the germ plasm and continuously propagable cancer cells escape from the inevitability of biological aging.—Hayflick, L. Current theories of biological aging. Federation Proc. 34: 9–13, 1975.

Protein synthesis directed by the RNA from a plant virus in a normal animal cell.

RNA from tobacco mosaic virus can be translated inside oocytes of the frog Xenopus laevis. The main product is a polypeptide with a molecular weight of 140,000. There is no evidence for coat protein synthesis, and it is unlikely that the polypeptide that is made contains either a whole or a partial coat protein sequence. The picture of translation of tobacco mosaic virus RNA obtained using oocytes is very much simpler than that found using cell-free protein-synthesizing systems, in which a great many polypeptides are made under the direction of tobacco mosaic virus RNA. The reasons for this difference are discussed, and the relative merits of in vivo and in vitro protein-synthesizing systems are compared.

A Restriction Point for Control of Normal Animal Cell Proliferation

This paper provides evidence that normal animal cells possess a unique regulatory mechanism to shift them between proliferative and quiescent states. Cells cease to increase in number under a diversity of suboptimal nutritional conditions, whereas a uniformity of metabolic changes follows these nutritional shifts. Evidence is given here that cells are put into the same quiescent state by each of these diverse blocks to proliferation and that cells escape at the same point in G(1) of the cell cycle when nutrition is restored. The name restriction point is proposed for the specific time in the cell cycle at which this critical release event occurs. The restriction point control is proposed to permit normal cells to retain viability by a shift to a minimal metabolism upon differentiation in vivo and in vitro when conditions are suboptimal for growth. Malignant cells are proposed to have lost their restriction point control. Hence, under very adverse conditions, as in the presence of antitumor agents, they stop randomly in their division cycle and die.

Immature and abnormal erythrocytes present in the normal, healthy spleen.

Spleens from healthy adult cats and dogs were perfused with oxygenated Ringer solution. The majority of the erythrocytes were quickly washed out but approximately 10 % were released very slowly with a half‐life of 1 hr (slow compartment). After perfusion the morphological characteristics of the cells in this slow compartment can be studied using histological sections prepared with blood stains. Under the light microscope a variety of immature and abnormal erythrocytes was seen. The immature cells were identified as rubricytes and reticulocytes, but no proerythroblasts were found. The pictures show rubricytes in various stages of maturation and denucleation evidently occurred by extrusion of either an intact or a fragmented nucleus. It is concluded that in the normal healthy animal cells released from the bone marrow at the prorubricyte stage undergo maturation in the spleen.

Mitochondrial oxidative phosphorylation.

AbstractMitochondria can form ATP from ADP and inorganic phosphate, the required energy being supplied by respiration. This coupled process, which in sufficiently aerated normal animal cells furnishes the bulk of the cellular ATP, is termed oxidative phosphorylation. The overall reaction is intimately associated with the mitochondrial inner membrane and proceeds via a primary high‐energy intermediate. This intermediate, in a manner as yet unknown, energizes the anhydride formation between ADP and inorganic phosphate. The coupling between respiration and ATP synthesis is mediated by proteins of the mitochondrial inner membrane which are known as “coupling factors”. The mechanism of oxidative phosphorylation is at present being discussed in terms of three hypotheses which are generally referred to as “chemical”, “chemiosmotic”, and “conformational” hypotheses. None of these hypotheses has as yet been experimentally verified.

Characterization of the time variations of reproductive processes in living cells.

SINCE the first published account of the duration of some phase in the mitotic cycle of living cells by Mitscherlich1 in 1848, there have been more than 800 publications on the in vivo and in vitro kinetics of normal and neoplastic animal cells. Implicit in most calculations, based on these observations and in mathematical models used in the analysis of the life cycle of mammalian cells, is the assumption that the generation time (that is, mitotic cycle time, replication time) is constant2–5, or so nearly so that variations can be ignored. The potential danger of assuming a mean or “average cell” in models of growth, normal and neoplastic cell intercomparisons, and chemotherapy schedules has not been generally recognized. Likewise, the assumption of a normal distribution or other distribution functions of mathematical convenience contributes little to our insight into the physical or biological process which results in a particular distribution of, for example, DNA replication time, cell duplication, time and mitotic time6. The danger of using mean times or even appropriate frequency functions to describe cell replication without knowing the magnitude of circadian rhythm in cell physiology7 and DNA synthesis rates8 has not been emphasized by most workers concerned with mammalian, normal and abnormal cell physiology and cancer chemotherapy. The great variability in cell replication time for individual cell types in constant environmental conditions is a biological fact of importance in understanding cell behaviour and effecting optimum chemotherapy. This communication presents a technique for characterizing cell replication times and associated variability. Previous attempts to graduate cell variability were initiated with the work of Rahn9, Kendall10, Powell11,12, and more recently Kubitschek13,14. About eight frequency functions which fit the distribution of generation time or the reciprocal of generation time, “generation rate”, have been presented. These functions fit the positively skewed bell-shaped curve with about the same degree of “good-ness-of-fit”6 but tell us little about the underlying biophysical processes. We, in common with Powell and Errington15, do not believe that a normal distribution can be used to describe the reciprocal of generation time advocated by some13,14,16. We have examined a model which not only characterizes quantitatively the kinetic parameters of cell growth but also allows inclusion of a circadian rhythm function. The derivation follows from the assumption that the number of cells which are dividing at time t is directly related to the time which has elapsed since some threshold, t0, which is necessary for the essential events to have occurred before replication. Assuming that the number of cells dividing is also directly proportional to the number of unfissioned cells of those which were originally present in the environment, then where k2 is some positive coefficient and N0 is the number of cells in the environment at t = 0. Furthermore, it is assumed that all N0 cells were newly born at t = 0. If k2 = 2α, then . The constant can be evaluated from initial conditions to get This equation gives a bell-shaped curve skewed to the right when (dN/dt) is plotted against time. It is important to note that there is no remarkable difference between the fit of this function and that of others10–12 to experimental data. The probability frequency function is This relation was fit to the experimental data on the bacterium, E. coli13; the “functional mitotic interval” from ascites tumour Epithelioma 25517; the protozoan Euglena gracilis18 in two environmental conditions; human amnion cells16, and HeLa cells19, using a method of least squares with computing machines. The results are shown in Fig. 1 as the cumulative frequency curves (integrated form of equation 2). Equation 2 has been evaluated for nineteen sets of experimental results of generation time measurements on a wide variety of cells and in all cases a good fit to the observed data was found (see Table 1), but it should be emphasized that many different distribution functions can be made to fit these experimental data. Furthermore, although the same function may fit all experimental data, one cannot argue that this implies that the same processes control the phenomena being observed—it is a necessary but not a sufficient condition.

Intermediates of Fatty Acid Metabolism by Cunninghamella Echinulata

The breakdown of fatty acids in animal tissues occurs by the /?oxidation route, in which oxidation proceeds by the removal of two carbon atoms at a time to yield acetyl-coenzyme-A and a fatty-acylcoenzyme-A two carbon atoms shorter than the initial compound. The history of investigations leading to our current understanding of this process has been reviewed in detail by Green (1954). Interest in the mechanism of fatty acid oxidation by fungi was first stimulated by the discovery that ketones were present in certain cheeses and edible oils attacked by fungi (Starkle, 1924). Acklin (1929) demonstrated that various fungi convert individual fatty acids into methyl ketones of one less carbon atom than the starting acid. Thaler et al. (1949) proposed the ^-oxidation pathway to account for the forma? tion of methyl ketones by fungi, the /?-keto acid formed by this process being decarboxylated to yield the methyl ketone. This proposal, up to the formation of the methyl ketone, agrees with current concepts of the process in animal cells. However, methyl ketones are not formed in appreciable amounts in normal animal cells, indicating the two systems are not identical.

The fate of rapidly labelled ribonucleic acid in the presence of actinomycin in normal and virus-infected animal cells

The rapidly labelled 35 to 45-S RNA of L cells is replaced during a chase period in the presence of actinomycin by 30-S and 20-S ribosomal sizes. This process takes place in the nucleus and most of the RNA remains within the nucleus for up to 4 h. At the same time some labelled 20-S, but not 30-S, RNA appears in the cytoplasm. In cells infected with encephalomyocarditis virus the amount of 32P found in nuclear 35 to 45-S RNA after a 30-min pulse quickly falls below that in normal cells. On adding actinomycin to stop further RNA synthesis the pulse-labelled RNA is not destroyed but is converted to 30-S and 20-S RNA just as in uninfected cells. Therefore the subnormal labelling after infection is probably due to inhibition of RNA synthesis rather than to an enhanced specific breakdown of rapidly synthesised RNA.

MALIGNANT TRANSFORMATION BY POLYOMA VIRUS.

Since the pioneering work of Rous. most research on tumor viruses has been carried out by oncologists rather than by virologists. However, with the recent rapid development of new knowledge concerning the basic mecha­ nisms involved in cytolytic infections with animal viruses, the attention of virologists has been turned to the same questions in the transformation of normal animal cells to malignant cells by the action of tumor viruses. Yet. even now, the amount of such research is limited chiefly because of a lack of available tumor virus-cell systems which lend themselves to modern molecu­ lar biological techniques. The polyoma virus first isolated and studied by Stewart et al. (1) has been investigated by a number of virologists for two reasons. First, it is a more interesting virus since it has the potential of producing a large variety of tumor types in its natural animal host [Stewart et al. (2)]; and, secondly because it can be studied quantitatively by modern virological methods. Although capable of causing malignant transformation in both in vivo and in vitro systems it nevertheless produces a cytolytic infection under certain conditions, and, therefore, can be quantitated in tissue cultures using the plaque technique [Dulbecco & Freeman (3)]. Furthermore, being able to agglutinate guinea pig red blood cells, it can be quantitated by hemagglutina­ tion and through the hemagglutination inhibition (HI)! test can be specifi­ cally identified with ease [Eddy et al. (4)). Since its natural host is the mouse, the availability of many susceptible inbred lines of laboratory mice make it possible to relate in vitro tissue culture phenomena to tumor production in the intact whole animal. This paper will review and evaluate the more important recent research contributions concerning mechanisms of polyoma virus transformation, the relationships between virus and the cell after transformation, and some of the virus-specific properties of the transformed cells. These items will first be considered for the �n vivo transformation and then for that occurring in in vitro systems.

Mitotic index in vitro of embryonic heart fibroblasts of different donor ages

Cultured normal human and animal cells are predestined to undergo irreversible functional decrements that mimick age changes in the whole organism. When normal human embryonic fibroblasts are cultured in vitro, 50 ± 10 population doublings occur. This maximum potential is diminished in cells derived from older donors and appears to be inversely proportional to their age. The 50 population doubling limit can account for all cells produced during a lifetime. The limitation on doubling potential of cultured normal cells is also expressed in vivo when serial transplants are made. There may be a direct correlation between the mean maximum life spans of several species and the population doubling potential of their cultured cells. A plethora of functional decrements occur in cultured normal cells as they approach their maximum division capability. Many of these decrements are similar to those occurring in intact animals as they age. We have concluded that these functional decrements expressed in vitro, rather than cessation of cell division, are the essential contributors to age changes in intact animals. Thus, the study of events leading to functional losses in cultured normal cells may provide useful insights into the biology of aging.

Le Auxine in Biologia Animale: Parte II - Azione Dell'acido Indolacetico e Della Kinetina in Vitro Su Tessuti Normali e Tumorali

The present paper is concerned with mitokinetic and cytokinetic action of two auxins — Kinetin and indolacetic acid — on «in vitro » cultures of normal animal cells (heart fibroblasts of chick embryo), and of neoplastic cells (from ascites Walker adenocarcinoma of the rat). The «in vitro » cultures were kept into contact with Kinetin doses ranging between 0.0005 and 0.001 γ/ml for normal tissues, and between 0.001 and 1 γ/ml for neoplastic cells. Indolacetic acid was used in doses ranging between 0.00005 and 0.0005 γ/ml for normal cells, and between 0.0005 and 0.05 γ/ml for neoplastic cells. Consideration has been given to the growth properties of the cultures, to the frequency of mitoses, and to the content in nucleic acids determined by the histochemical method and by the orange-acridin in U.V. light method. It has been demonstrated that Kinetin, even at low doses, exerts an inhibitory action on the heart fibroblasts growth, with considerable cytitoxic action and partial inhibition of the mitotic activity. Indolacetic acid did not show any cytotoxic action, and failed to demonstrate any cyto- and mitokinetic activity. Experiments carried out with the association of both auxins in suitable dosage showed a prevailing cytotoxic action of Kinetin, and were not able to demonstrate a mitotic activity. In addition, the single or associate administration of the two auxins did not influence the DNA synthesis in normal cells. In «in vitro» cultures of cells from ascites Walker adenocarcinoma an early and constant mitokinetic activity has been demonstrated by Kinetin and indolacetic acid as well. Cytotoxic activity was absent, even at high concentrations. No synergic action was shown by the two auxins.

Contribution cytochimique et histoautoradiographique à l'étude du métabolisme et de la synthése des acides d́esoxyribonucléiques dans des cellules animales cultivées in vitro—I : Études cytophotométrique et histoautoradiographique des ADN dans des fibroblastes traités par le Myleran

We have studied the behaviour of normal animal cells cultivated in vitro in the presence of Myleran for variable periods ranging from a few hours to 18 days. The cytological modifications produced by this substance have already been described previously. The present report deals essentially with the study of DNA which we have carried out by means of quantitative cytophotometry of individual cells after Feulgen reaction as well as by histoautoradiography after the incorporation of tritiated thymidine. The Myleran profoundly modifies the metabolism of DNA. It provokes an increase of positive Feulgen material in the interkinetic nuclei. This effect becomes more and more marked with time: nuclei with tetraploid value are more frequent, octoploid and even hexadecaploid nuclei appear and become progressively more numerous. Nevertheless, it does not appear that the number of genoms increases, in spite of the possibility of eventual fragmentation, The frequency of mitosis is very low. Under the influence of Myleran there is thus a dissociation between the frequency of tetraploid values of DNA and that of the mitoses. In cultures having been treated for a sufficient time, the cytophotometric estimations of mitotic figures (two wavelength method) show that a certain proportion of mitoses: prophases, metaphases, etc., reach octoploid DNA values, contrary to what happens in the controls. Fibroblasts having this peculiarity are thus capable of undergoing mitosis and to complete it. The histoautoradiographies clearly show that tritiated thymidine is incorporated by the nuclei even if the mitotic activity has been reduced. The level of this incorporation is related to the increase of frequency of nuclei with high DNA content. The cells therefore remain able to synthesize DNA even after long action of Myleran. A hypothesis is proposed to explain the observed facts. According to this, the cell after each mitosis, synthesizes a fresh quantity of deoxyribonucleic acids to compensate for those which have been affected by the Myleran. It will then undertake the preprophase synthesis in readiness for the subsequent mitosis by doubling the quantity of DNA then present in the nucleus. In this way the amount of DNA in the nuclei will increase in time. In addition, under certain aspects, our experiments suggest that the synthesis of DNA does not take place following classical concepts (i.e. from a “model”, by duplication of existing material) but following a different process, from a “plan”.

Effects of kinetin on cell division in Paramecium caudatum.

KINETIN (6-furfurylaminopurine) has been reported to stimulate cell division in excised tobacco pith tissues1, in onion root tips2 and, most recently, in sarcoma tumour cells3. Lettre and Endo4 found no changes in cell division or tumour growth with kinetin in cultures of normal or malignant human and animal cells. They concluded that these tissues either did not possess a kinetin-like trigger for cell division, or contained optimal levels of such a substance.

Uptake of Benzpyrene by a Chromosomal Phospholipid

THE uptake of 3,4-benzpyrene by cells has been of interest to many workers, particularly in view of the carcinogenic properties of this substance. Moreover, it is a fluorescent compound and has been used as a fluorochrome for lipids into which it will dissolve from a hydrotropic solution ; caffeine is usually used for dispersing it finely in water, since alone benzpyrene appears to be almost completely hydrophobic. Berg1 has used this method in a detailed and extensive study of the distribution of lipids in a wide range of normal and cancerous animal cells and in the root tips of Allium cepa. In order that the lipids should not be affected by his preparatory procedures, he fixed the tissues in formalin which had been stored over powdered chalk ; chalk seems to have the same effect as calcium chloride, which is frequently added with the intention of maintaining a high concentration of calcium ions to immobilize lipids2. This method of fixation seems to be much favoured by histologists. The tissue was sectioned with a freezing microtome, and the sections were then stained with benzpyrene. After a very careful investigation, Berg was forced to conclude that in animal and plant nuclei lipids are not present except in the nuclear membrane, and that biochemical demonstrations of these compounds in extracted nuclei were due to contamination by the cytoplasm. Indeed, he found that the nucleus was the one region in cells that never showed uptake of benzpyrene.

Further Comments on the Causation of Malignant Disease

Invisible infective agents may be divided into: (1) true, ultramicroscopic, living viruses, which do not arise de novo and, so far as is known, are not ubiquitous; (2) transmissible infective agents which arise de novo and are propagated through living cells, but are not themselves living organisms; (3) stimulants to variation which arise de novo, are not transmissible, and are not living organisms.Class (1) is not represented in malignant disease. “Bacteriophage” is a representative of class (2); very probably the infective agent of fowl sarcoma comes under the same category, and possibly some important human diseases of doubtful aetiology. There is no satisfactory evidence that mammalian malignant disease is related to class (2); its causation, according to the “chronic irritation” theory, must be attributed to influences comprised within class (3).The stimulants to variation in class (3) depend for their effectiveness upon the unstable energy of living matter. The changes which they produce are “biological” in the sense that they are changes of chemical constitution which could not be obtained without the aid of vital processes.Regulation of normal growth in the animal body means regulation of the cell's facilities for obtaining energy. I think it is misleading to regard it as a forceful restraint (or stimulus) upon the cell's inherent capacity for unlimited growth.The assumption, borrowed from “natural immunity” towards bacteria, that there is in the animal body a natural principle which destroys frequently occurring foci of incipient malignancy is also unsubstantiated and misleading.During the latent period, certain cells, which subsequently grow into a neoplasm, lose their capacity to respond to inhibitory systemic influences. This change is brought about by local and not by systemic causes.As regards the special class of tumour derived from cells which have been displaced in foetal life, long residence in an abnormal situation does not appear to be equivalent to the ordinary latent period; but it may have had the effect of increasing their susceptibility, so that, if exposed to chronic irritation, the cells would more readily lapse into the latent period predisposing to malignancy.It is known that the various tissues of the animal body differ in their degree of susceptibility to the precancerous change. This is a cellular characteristic; so also is the difference in the susceptibility of one animal as compared with another. It is not a question of difference in a hypothetical humoral property of “systemic resistance.”On the termination of the latent period by a fresh stimulus to proliferation, certain cells commence active growth and are incapable of responding to systemic inhibitory influences. These conditions seem sufficient for the origin of an innocent neoplasm. But something more is required to explain malignancy, because the malignant cell is essentially different from the cells in a benign tumour.About the actual cause of the change to malignancy one can only offer conjectures. I have suggested a way in which the change may possibly be produced through the agency of the local endothelium and the autogenous formation of antibodies.On taking a broad view, the change into the malignant variant is not something unique; equally remarkable changes are to be found in the properties of bacteria. In both cases the facts have to be accepted, at present, without satisfactory explanation of the conditions which gave rise to them. One finds with bacteria that degradation or “roughness” may be a phase preparatory to the acquirement of new properties, just as the degradation of cells in the latent period seems to be a requisite preparation for acquiring the new property of malignancy. But the actual steps involved in the change from a bacterial saprophyte to an invasive parasite are as difficult to understand as are the processes involved in the conversion of a normal animal cell into its malignant variant.Throughout the study of cancer it is very desirable to maintain a clear distinction between cause and effect. For example, the enzymes peculiar to cancer are not the cause of cancer but the effect of the biological change which produced the cancerous cell.