PubMed Health⌕ Search

Biomedical subjects

Harry Rubin

Publications and source records attributed to Harry Rubin.

15 recordsLinked to original sources
PubMed2006

The logic of the Membrane, Magnesium, Mitosis (MMM) model for the regulation of animal cell proliferation.

The addition of animal serum or specific protein growth factors to quiescent, serum-deprived vertebrate cells in culture activates a wide variety of biochemical responses within minutes, which are followed in 5-10h by the initiation of DNA synthesis and then by mitosis. The quintessential early and continuing activation step for the increase in DNA synthesis is the increased initiation rate of protein synthesis, which must be continuously maintained throughout the G1 phase for advancement into S. The aggregate of biochemical reactions to growth factors is called the coordinate response, to indicate that many related and unrelated processes are orchestrated to repetitively reproduce cells. It is, however, crucial to recognize that the coordinate response can be induced for one or more rounds of replication by a variety of non-specific and quasi-specific membrane effectors. The logic of considering this framework of events in growth control implied that a single multi-target second messenger plays a central role in coordinating the events of the overall response. The same reasoning suggested that free Mg(2+) is the unifying regulatory element in that response which includes protein kinase pathways, and that the cytoplasmic activity of Mg(2+) increases with the binding of growth factors to their receptors in the cell membrane, or of less specific perturbations of the membrane. Experimental support of this conclusion is presented here and is represented in the MMM model of cell proliferation control.

Animals↗
PubMed2006

What keeps cells in tissues behaving normally in the face of myriad mutations?

The use of a reporter gene in transgenic mice indicates that there are many local mutations and large genomic rearrangements per somatic cell that accumulate with age at different rates per organ and without visible effects. Dissociation of the cells for monolayer culture brings out great heterogeneity of size and loss of function among cells that presumably reflect genetic and epigenetic differences among the cells, but are masked in organized tissue. The regulatory power of a mass of contiguous normal cells is expressed in its capacity to normalize the appearance and growth behavior of solitary homophilic neoplastic cells, and to redirect differentiation of solitary heterophilic stem-like cells. Intimate contact between the interacting cells is required to induce these changes. The normalization of the neoplastic phenotype does not require gap junctional communication between cells, though transdifferentiation might. These varied relationships are manifestations of the unifying biological principle of "order in the large over heterogeneity in the small".

Aging↗
PubMed2005

Systemic effects of cancer: role of multiple proteases and their toxic peptide products.

Cachexia, or the loss of skeletal muscle, adipose tissue and immunological competence, is a common systemic condition in advanced cases of cancer, and is a frequent cause of death. There have been many reports that small peptides isolated from tumors induce systemic effects correlated with cachexia upon injection into mice or addition to cell cultures. Only recently has evidence been presented for the origin of these peptides from the large increase in proteolytic enzymes found in tumors. Additional evidence is presented here for a major role of tumor peptides in producing effects associated with cachexia. Such peptides of low molecular weight were found to produce lipolysis in adipose tissue. Fibrinogen degradation products from the plasma of cancer patients and other chronic diseases suppress activation of the immune response. Some proteases of the cathepsin class occur in high concentration at the surface of transformed cells where the pH is low enough to allow digestion of pericellular proteins; other cathepsins elevated in tumors are highly active at physiological pH. Cathepsin L is the major excreted protein of cultured cells transformed by viruses or chemicals and is likely to form toxic peptides by digestion of extracellular proteins. Even normal liver and skeletal muscle of some cancer patients and tumor-bearing animals exhibit significant increases in cathepsins and components of the ubiquitin-proteasesome pathway which would add to circulating peptides. These observations argue for a central role of multiple proteases and their proteolytic products in producing the debilitating systemic effects of cancer.

Adipose Tissue↗
PubMed2005

Degrees and kinds of selection in spontaneous neoplastic transformation: an operational analysis.

Spontaneous neoplastic transformation develops within days in the NIH 3T3 line of cells through differential inhibition of their proliferation under contact inhibition. A small fraction of the population continues to multiply after saturation density is reached and is selected to progressively increase saturation density in successive rounds of confluence. The degree of selection at confluence depends on the extent of proliferation of some cells in a heterogeneous population. The development of transformed foci is an extension of the same selective process that increases saturation density. The expression of the foci is enhanced with increases in the saturation density of the surrounding cells. Transformation is also induced by moderately reducing the concentration of calf serum in the medium during low-density passages, which allows selection of cells that require less growth factor. Further stepwise reductions in serum increase the degree of transformation. Contact inhibition and reduction in serum concentration select for the same phenotype of cell that increases saturation density and generates transformed foci. There is mounting evidence that selection is a major factor in the development of common epithelial tumors of humans, but it extends over decades rather than days, and the in vivo microenvironment selects from more stable populations of cells than those in culture. The many progressive levels of increased saturation density and transformed focus formation suggest that a very large number of genes participate in neoplastic development. The operational model of variation and selection presented here may aid in understanding chemical carcinogenesis and cancer recurrence after chemotherapy.

Animals↗
PubMed2005

Mg2+ as activator of uridine phosphorylation in coordination with other cellular responses to growth factors.

The divalent cation ionophore A23187 facilitates the manipulation of intracellular Mg2+ without increasing the general permeability of the cell. The uptake of uridine into cells is limited by its rate of intracellular phosphorylation that increases within minutes after the addition of growth factors. In the experiments described here, the rate of uridine uptake in ionophore-treated cells stimulated by either serum or insulin depended on the extracellular and intracellular concentrations of Mg2+ and was independent of the extracellular Ca2+ concentration. In very high concentrations of Mg2+ (50 mM), ionophore-treated cells take up uridine as fast, in the absence of growth factors as in their presence, demonstrating that Mg2+ can replace the growth factor requirement for the stimulation of uridine uptake. In contrast, thymidine uptake, which also is limited by its rate of intracellular phosphorylation, showed no early response to either growth factors or Mg2+ concentration, which is consistent with the 10-fold lower Mg2+ requirement of thymidine kinase compared with uridine kinase. The feedback inhibition of uridine kinase by UTP and CTP in cell-free extracts was alleviated by increased Mg2+ concentration. The results support the thesis that the increased uptake of uridine in cells treated with growth factors is determined by a membrane-induced increase in intracellular free Mg2+. Such increase would also accelerate the rate of translation-initiation and other coordinate responses that, unlike increased uridine uptake, are essential for cell proliferation. The rate of uridine uptake is suggested as a direct indicator of free cytosolic Mg2+ that drives the shift from quiescence to proliferation.

3T3 Cells↗
PubMed2005

Magnesium: The missing element in molecular views of cell proliferation control.

The quantitative study of regulation of cell growth and proliferation began with the development of the technique for monolayer culture of vertebrate cells in the late 1960s. The basic parameters were defined in the early physiological studies, which continued through the next decade. These included specific and non-specific growth factors and the requirement for continuous exposure to such factors through most of the G1 period for progression to S. In the course of this work, the diversity of biochemical responses and the critical role of increased protein synthesis and accumulation for the onset of DNA synthesis were elucidated. In particular, a central role of free cytosolic Mg2+ in direct regulation of protein synthesis and in ancillary processes as a response to membrane perturbation was established. Eventually, the physiological era was superseded by the molecular era beginning in the 1980s. This work focussed on specific receptors for growth factors that entrained a protein kinase cascade, which terminated in a higher frequency of initiation of protein synthesis. However, the molecular studies virtually ignored the key results of the physiological era. Recent studies of the penultimate molecular steps in the regulatory pathway of protein synthesis, however, have supported a model of growth regulation involving membrane perturbation and MgATP2- concentration, results that integrate the findings of the physiological and molecular eras. The resulting relatively simple "membrane, magnesium mitosis" (MMM) model of proliferation control can explain the seeming paradox of the variety of specific and non-specific growth-enhancing treatments that are mediated by the plasma membrane and which bring about a shared, complex but coordinated growth response that drives cell proliferation.

Adenosine Triphosphate↗
PubMed2005

Membrane stabilization by intimate contact between cells reduces their Mg2+ activity and suppresses the neoplastic phenotype.

The mechanism of normalizing transformed cells by contact with normal cells is considered in relation to the mechanism of stimulating the growth of normal contact-inhibited cells. Stimulation is based on perturbation of the plasma membrane throughout the G1 period by growth factors. A primary effect of the membrane perturbation is a long-sustained increase of intracellular free Mg(2+) which increases the initiation frequency of polypeptide synthesis and accelerates the onset of DNA synthesis. Because the plasma membrane of transformed cells is kept in a continuously perturbed state and forms poor intercellular adhesions, it loses control of intracellular Mg(2+) activity. However, maximizing contact between transformed cells at extremely high density stabilizes their plasma membrane, lowers their Mg(2+) content and normalizes their phenotype. Normalization may also be obtained in contact with lower densities of quiescent, confluent normal cells. Evidence against a need for junctional communication supports membrane stabilization with restoration of Mg(2+) regulation as the mechanism of normalization.

Animals↗
PubMed2005

Central roles of Mg2+ and MgATP2- in the regulation of protein synthesis and cell proliferation: significance for neoplastic transformation.

Growth factors are polypeptides that combine with specific membrane receptors on animal cells to stimulate proliferation, but they also stimulate glucose transport, uridine phosphorylation, intermediary metabolism, protein synthesis, and other processes of the coordinate response. There are a variety of nonspecific surface action treatments which stimulate the same set of reactions as the growth factors do, of which protein synthesis is most directly related to the onset of DNA synthesis. Mg(2+) is required for a very wide range of cellular reactions, including all phosphoryl transfers, and its deprivation inhibits all components of the coordinate response that have so far been tested. Growth factors raise the level of free Mg(2+) closer to the optimum for the initiation of protein synthesis. The resulting increase in protein synthesis accelerates progression through G1 to the onset of DNA synthesis and mitosis. None of the other 3 major cellular cations are similarly involved in growth regulation, although internal pH may play an auxiliary role. Almost 10(5) externally bound divalent cations are displaced from membranes for every attached insulin molecule, implying a conformational membrane change that releases enough Mg(2+) from the internal surface of the plasma membrane to account for the increase in free cytosolic Mg(2+). It is proposed that mTOR, the central control point for protein synthesis of the PI 3-K kinase cascade stimulated by insulin, is regulated by MgATP(2-) which varies directly with cytosolic Mg(2+). Other elements of the coordinate response to growth factors such as the increased transport of glucose and phosphorylation of uridine are also dependent upon an increase of Mg(2+). Deprivation of Mg(2+) in neoplastically transformed cultures normalizes their appearance and growth behavior and raises their abnormally low Ca(2+) concentration. Tight packing of the transformed cells at very high saturation density confers the same normalizing effects, which are retained for a few days after subculture at low density. The results suggest that the activity of Mg(2+) within the cell is a central regulator of normal cell growth, and the loss of its membrane-mediated control can account for the neoplastic phenotype.

Adenosine Triphosphate↗
PubMed2003

Cancer cachexia: its correlations and causes.

Cancer cachexia involves the loss of weight, mainly in skeletal muscle and adipose tissue, that is not caused simply by anorexia. The syndrome includes anemia and immunosuppression along with a number of biochemical changes indicating systemic effects of the cancer. It is a major factor in morbidity and mortality from cancer. For 30 years beginning in 1948, a large number of studies reported isolation from many tumors of a heterogeneous group of small peptides, generally labeled toxohormone, that caused various correlates of cachexia shortly after injection into mice. Interest in toxohormone-like peptides then fell off for diverse reasons that had little to do with their clinical significance and was shifted to cytokines, ILs, and ectopic hormones with catabolic consequences that were sporadically found in tumors. At the same time, evidence was accumulating for an important role of pericellular proteases in driving progressive stages of neoplastic development. A central part of that evidence was the inhibition of transformation-related changes by protease inhibitors, particularly the combination present in fetal bovine serum, which fully suppressed the expression of the transformed phenotype in discrete foci of chicken embryo fibroblasts (CEF) infected by Rous sarcoma virus against a confluent background of uninfected CEF. In contrast, CEF cultures heavily infected with Rous sarcoma virus in the same medium underwent pervasive transformation, which was correlated with the release of low molecular weight cytotoxic substances. Reevaluation of all of the evidence supports a central role for proteolytically generated peptides derived from tumors in producing cancer cachexia.

Cachexia↗
PubMed2003

Microenvironmental regulation of the initiated cell.

In the classical skin model of tumor initiation, keratinocytes treated once with carcinogen retain their normal appearance and growth behavior indefinitely unless promoted to growth into papillomas. Because many of the papillomas regress and may recur with further promotion, their cells can also be considered as initiated. The growth of initiated keratinocytes can be inhibited either in vitro or in vivo by close association with an excess of normal keratinocytes, but it is enhanced by dermal fibroblasts. Chick embryo fibroblasts (CEF) in culture produce transformed foci after infection with Rous sarcoma virus (RSV) on a background of normal CEF in a medium containing 10% or less calf serum (CS), but they retain normal appearance and growth regulation in 10% fetal bovine serum (FBS) or 20% CS. Transformation of a carcinogen-treated line of mouse embryo fibroblasts is prevented, and can be reversed, in high concentrations of FBS in the presence of an excess of normal cells. FBS has high, broad-spectrum antiprotease activity. Increased protease production occurs in a variety of transformed cells and is correlated with progression in tumors. Protease treatment stimulates DNA synthesis and mitosis in confluent, contact-inhibited normal cell cultures. Synthetic inhibitors of proteases suppress transformation in carcinogen-treated cultures and inhibit tumor formation in animals. Several different classes of protease may be overexpressed in the same transformed cells. It is proposed that excessive protease production accounts for major features of neoplastic transformation of initiated cells, but that transformation can be held in check by protease inhibitors present in serum and released from surrounding cells. It would be informative to determine whether high concentrations of FBS would inhibit the neoplastic development of initiated keratinocytes.

Animals↗
PubMed2003

Complementary approaches to understanding the role of proteases and their natural inhibitors in neoplastic development: retrospect and prospect.

A great deal of evidence has accumulated in recent years for an important but complex role for proteases in tumor development. However, attempts to treat cancer in humans with anti-proteases have been disappointing, and it has been suggested that more basic groundwork is needed before anti-proteases can be effectively applied. Considerable basic information comes from the recognition that earlier results on transformation of chicken embryo fibroblasts (CEF) by the Bryan strain of Rous sarcoma virus (B-RSV) can be explained in terms of proteases and their inhibitors. In particular, the full but reversible normalization of discrete transformed foci by appropriate concentrations of fetal bovine or of calf serum implies a causal role for multiple proteases in transformation, and the efficacy of treatment with a physiological balance of their natural inhibitors. Addition of certain proteases to contact-inhibited normal cultures was then found to stimulate their proliferation. The toxicity of medium produced by CEF heavily transformed with B-RSV suggests that cachexia and other systemic effects of human cancer may result from vascular dissemination of peptides from pericellular proteolysis within tumors. Comprehensive studies revealed significant increases of plasminogen activator and matrix metalloproteinases (MMPs) after infection of CEF with other strains of RSV, and correlation of the proteases with aspects of transformation. A similar role for proteases is indicated in the transformation of mammalian cells by chemical and physical agents. The information gained from functional experiments on cell transformation in culture is complementary to that obtained from the molecular identification of proteases and their inhibitors in all stages of tumor development. The speed, quantification and easy manipulation of the RSV-CEF transformation assay can be combined with current methods of characterizing proteases and anti-proteases to further enrich our basic knowledge of neoplastic development in cells, and facilitate its application to the treatment of cancer.

Animals↗
PubMed2002

Selective clonal expansion and microenvironmental permissiveness in tobacco carcinogenesis.

Historically our knowledge about the direct carcinogenic activity of cigarette smoke and its constituents grew from painting experiments on the skin of mice to produce papillomas and carcinomas. The neutral fraction of cigarette smoke condensate had most of the carcinogenic activity in this test and was rich in carcinogenic polycyclic aromatic hydrocarbons (PAHs), the most abundant by far being BP. However, the concentration of BP in the condensate was only about 2% the amount of pure BP required to cause skin tumors. In other fractions there were non-carcinogenic constituents that promoted tumor formation when applied repeatedly to mouse skin that had been initiated by a single subcarcinogenic application of BP. There were also constituents of cigarette smoke that acted as co-carcinogens when applied simultaneously with repeated applications of BP. BP was effective as an initiator at lower concentrations than as a complete carcinogen, and some non-carcinogenic PAHs in the condensate were also active initiators. It was concluded from these studies that cigarette smoke condensate is primarily a tumor-promoting and co-carcinogenic agent with weak activity as a complete carcinogen. A major effect of promoters, and possibly of co-carcinogens, is a diffuse hyperplasia which includes selective expansion of clones carrying endogenous mutations and/or mutations induced by PAHs and other carcinogens such as NNK. The induced mutations as well as damaged cells would occur throughout the exposed region and, along with the hyperplasia, increase the permissiveness of the cellular microenvironment for neoplastic expression of any potential tumor cell in its midst. Since neither the promoters nor co-carcinogens in tobacco smoke are known to interact directly with DNA, their effects can be considered epigenetic processes that act upon genetically altered cells. Examples are cited from studies of experimental skin carcinogenesis, smoking-induced histopathological changes in human lung and spontaneous transformation in cell culture to illustrate the genetic and epigenetic interactions of neoplastic development in general and their significance for smoking-induced lung cancer in particular. Certain dietary modifications that appear to be effective in moderating the promotional phase of animal and human carcinogenesis are suggested for trial in managing lung cancer.

Animals↗
PubMed2002

The disparity between human cell senescence in vitro and lifelong replication in vivo.

Cultured human fibroblasts undergo senescence (a loss of replicative capacity) after a uniform, fixed number of approximately 50 population doublings, commonly termed the Hayflick limit. It has been long known from clonal and other quantitative studies, however, that cells decline in replicative capacity from the time of explantation and do so in a stochastic manner, with a half-life of only approximately 8 doublings. The apparent 50-cell doubling limit reflects the expansive propagation of the last surviving clone. The relevance of either figure to survival of cells in the body is questionable, given that stem cells in some renewing tissues undergo >1,000 divisions in a lifetime with no morphological sign of senescence. Oddly enough, these observations have had little if any effect on general acceptance of the Hayflick limit in its original form. The absence of telomerase in cultured human cells and the shortening of telomeres at each population doubling have suggested that telomere length acts as a mitotic clock that accounts for their limited lifespan. This concept assumed an iconic character with the report that ectopic expression of telomerase by a vector greatly extended the lifespan of human cells. That something similar might occur in vivo seemed consistent with initial reports that most human somatic tissues lack telomerase activity. More careful study, however, has revealed telomerase activity in stem cells and some dividing transit cells of many renewing tissues and even in dividing myocytes of repairing cardiac muscle. It now seems likely that telomerase is active in vivo where and when it is needed to maintain tissue integrity. Caution is recommended in applying telomerase inhibition to kill telomerase-expressing cancer cells, because it would probably damage stem cells in essential organs and even increase the likelihood of secondary cancers. The risk may be especially high in sun-exposed skin, where there are usually thousands of p53-mutant clones of keratinocytes predisposed to cancer.

Animals↗
PubMedDate not supplied

Promise and problems in relating cellular senescence in vitro to aging in vivo.

According to the 'Hayflick limit', human fetal fibroblasts have a uniform, limited replicative lifespan of about 50 population doublings in cell culture. This concept was extrapolated to diverse cells in the body. It seemed to decrease with the age of the cell donor and, as a form of cell senescence, was thought to underlie the aging process. More discriminating analysis, however, showed that the fibroblasts decayed in a stochastic manner from the time of their explantation, at a rate that increased with the number of population doublings in culture. There was no consistent relation to the age of the donor. Despite the contradictory evidence, the original version of the Hayflick limit retained its general acceptance. Cell senescence was attributed to the absence of telomerase in the fibroblasts, which resulted in shortening of telomeres at each division until they fell below a critical length needed for further division. However, it is well established that stem cells in renewing tissues undergo many more than 50 divisions in a lifetime, without apparent senescence. Contrary to early findings of no telomerase in most tissues, their stem cells retain telomerase and presumably telomere length despite many divisions in vivo. Massive accumulation of lipofuscin granules occurs under stress in long term crowded cultures, but the granules dissipate on subculture or neoplastic transformation. The overall results indicate a critical disjunction between cell senescence in vitro and aging in vivo. By contrast, cell culture has been useful in showing a need for telomere capping in maintaining cell stability and viability. It may also provide information about the biochemical mechanism of lipofuscin production.

Journal Article↗