Acoustic regularities in infant-directed speech and song across cultures.

When interacting with infants, humans often alter their speech and song in ways thought to support communication. Theories of human child-rearing, informed by data on vocal signalling across species, predict that such alterations should appear globally. Here, we show acoustic differences between infant-directed and adult-directed vocalizations across cultures. We collected 1,615 recordings of infant- and adult-directed speech and song produced by 410 people in 21 urban, rural and small-scale societies. Infant-directedness was reliably classified from acoustic features only, with acoustic profiles of infant-directedness differing across language and music but in consistent fashions. We then studied listener sensitivity to these acoustic features. We played the recordings to 51,065 people from 187 countries, recruited via an English-language website, who guessed whether each vocalization was infant-directed. Their intuitions were more accurate than chance, predictable in part by common sets of acoustic features and robust to the effects of linguistic relatedness between vocalizer and listener. These findings inform hypotheses of the psychological functions and evolution of human communication.

Deprivation of glutamine in cell culture reveals its potential for treating cancer.

The growth-stimulating capacity of calf serum (CS) in cell culture reaches a maximum of 10% with Balb 3T3 cells, remains at a plateau to 40% CS, and declines steeply to 100% CS. Growth capacity can be largely restored to the latter by a combination of cystine and glutamine. Glutamine is a conditionally essential amino acid that continues to function at very low concentrations to support the growth of nontransformed cells, but transformed cells require much larger concentrations to survive. These different requirements hold true over a 10-fold variation in background concentrations of CS and amino acids. The high requirement of glutamine for transformed cells applies to the development of neoplastically transformed foci. These observations have given rise to a novel protocol for cancer therapy based on the large difference in the need for glutamine between nontransformed and transformed cells. This protocol would stop the cumulative growth and survival of the transformed cells without reducing the growth rate of the nontransformed cells. The results call for studies of glutamine deprivation as a treatment for experimental cancer in rodents and clinical trials in humans.

Phenotypic selection as the biological mode of epigenetic conversion and reversion in cell transformation.

Exposure of certain cell lines to methylcholanthrene, X-rays, or physiological growth constraint leads to preneoplastic transformation in all or most of the treated cells. After attaining confluence, a fraction in those cells progress to full transformation, as evidenced by their ability to form discrete foci distinguishable from the surrounding cells by virtue of their higher density. Transformation induced by suspension in agar, an even stronger growth-selective condition than confluence, is reminiscent of all but the final differentiated stage of a normal developmental process, epithelial-mesenchymal transition. Changes associated with transformation are not restricted to focus-forming cells, as the permissiveness for focus formation provided by confluent cells surrounding transformed foci is greater than that of nonselected cells. The neoplastic process can also be reversed in culture. Transformed cells passaged at low density in high serum revert to normal morphology and growth behavior in vitro and lose the capacity for tumor formation in vivo. We propose that transformation and its reversal are driven by a process of phenotypic selection that involves entire heterogeneous populations of cells responding to microenvironmental changes. Because of the involvement of whole cell populations, we view this process as fundamentally adaptive and epigenetic in nature.

Dynamics of cell transformation in culture and its significance for tumor development in animals.

NIH 3T3 cells grown in conventional Dulbecco's modification of Eagle's basal medium (DME) produce no transformed foci when grown to confluence in 10% calf serum (CS). A few cultures were transformed by ras oncogenes when transfected with DNA from neoplastic cells, but they failed to do so in 80 to 90% of the transfections. However, when they were grown in a medium [molecular, cellular, and developmental biology 402 (MCDB 402)] optimized for their clonal growth in minimal serum, they produced transformed foci without transfection in 10% CS, but not in 2% CS. The first response to growth in MCDB 402 in 2% CS in successive rounds of contact inhibition was uniform increases in saturation density of the population. This was followed by the appearance of transformed foci. A systematic study was made of the dynamics of neoplastic progression in various concentrations of CS in a single round of confluence at 2 and 3 wk, followed by three sequential rounds of confluence in 2% CS for 2 wk. There was a linear relationship between CS concentration and saturation density in the first-round cultures and continuing differences in subsequent cultures. The hyperplastic field of normal-looking cells surrounding transformed foci became increasingly permissive for transformation with serial culture. The dynamics show that epigenetic selection is the major driving force of neoplastic development. Cells from dense foci produced malignant fibrosarcomas in mice, thereby exhibiting a positive relationship between transformation in culture and the development of tumors.

The paradox of the contrasting roles of chronic magnesium deficiency in metabolic disorders and field cancerization.

Magnesium (Mg(2+)) deficiency is common in metabolic disorders such as obesity, type 2 diabetes, and insulin resistance. These disorders are also associated with a high incidence of cancer. Mg(2+) is the regulator par excellence of metabolism, largely through its role as a cofactor for all phosphoryl transfers in the cell. Because Mg(2+) deficiency inhibits energy production it might be expected to inhibit tumor production. However, the high incidence of cancer in metabolic disorders makes that seem unlikely. In order to understand this seeming paradox, it is important to understand the regulatory role of Mg(2+) in normal and neoplastic cells. Free Mg(2+) is the primary regulator of glycolysis and the Krebs cycle. It also acts as a second messenger for growth factors in regulating protein synthesis. Varying Mg(2+) concentrations result in the same set of coordinated responses as varying serum concentrations. Selection by serial rounds of high cell density or reduced serum concentration at low cell density results in progressive stages of field cancerization. Highly transformed cells proliferate in much lower concentrations of Mg(2+) and grow to much higher saturation densities than normal cells. It remains to be seen whether reduction in Mg(2+) in sparse, exponentially proliferating cultures selects for increases in saturation density and transformed foci.

Promotion and selection by serum growth factors drive field cancerization, which is anticipated in vivo by type 2 diabetes and obesity.

Individuals suffering from type 2 diabetes or obesity exhibit a significant increase in the incidence of various types of cancer. It is generally accepted that those conditions arise from overnutrition and a sedentary lifestyle, which lead to insulin resistance characterized by overproduction of insulin acting as a growth factor. There is a consensus based largely on epidemiological data that chronic overproduction of insulin is responsible for the increased incidence of cancer. A model system in culture of NIH 3T3 cells induces the collective effects of serum growth factors on progression through the stages of field cancerization. It shows that the driving force of progression is promotion of cell growth under selection at high cell density, with no requirement for exogenous carcinogenic agents. The early effect is gradual selection among many preexisting, low-penetrance preneoplastic mutations or stable epigenetic variants, followed by sporadic, high-penetrance transforming variants, all dependent on endogenous processes. The significance of the results for cancer in diabetic and obese individuals is that the initial stages of the process involve multiorgan metabolic interactions that produce a systemic insulin resistance with chronic overproduction of insulin and localized field cancerization. Hypomagnesemia is prevalent in the foregoing metabalo/systemic disorders, and may also provide a selective microenvironment for tumor development.

The early history of tumor virology: Rous, RIF, and RAV.

One hundred years ago Peyton Rous recovered a virus, now known as the Rous sarcoma virus (RSV), from a chicken sarcoma, which reproduced all aspects of the tumor on injection into closely related chickens. There followed recovery of causal viruses of tumors of different morphology from 4 more of 60 chicken tumors. Subsequent studies in chickens of the biology of the first RSV isolated moved slowly for 45 y until an assay of ectodermal pocks of the chorioallantoic membrane of chicken embryos was introduced. The inadequacies of that assay were resolved with the production of transformed foci in cultures of chicken fibroblasts. There followed a productive period on the dynamics of RSV infection. An avian leukosis virus (ALV) was found in some chicken embryos and named resistance-inducing factor (RIF) because it interferes with RSV. Its epidemiology in chickens is described. Another ALV was found in stocks of RSV and called Rous-associated virus (RAV). Cells preinfected with RAV interfere with RSV infection, but RSV does not produce infectious virus unless RAV is added during or after RSV infection. Intracellular RAV provides the infectious coat for the otherwise defective RSV. The coat determines the antigenicity, host range, and maturation rate of RSV. RSV particles carry reverse transcriptase, an enzyme that converts their RNA into DNA and allows integration into the cell's DNA, where it functions as a cellular gene. This was the bridge that joined the biological era to the molecular era. Its relation to oncogenes and human cancer is discussed.

Fields and field cancerization: the preneoplastic origins of cancer: asymptomatic hyperplastic fields are precursors of neoplasia, and their progression to tumors can be tracked by saturation density in culture.

Most basic research on cancer concerns genetic changes in benign and malignant tumors. Yet evidence indicates that the majority of the mutations in tumors occur in the preneoplastic field stage of their development. That early stage is represented by grossly invisible, broad regions of "field cancerization" which have not, heretofore, been operationally analyzed in cell culture. Conditions are described for quantitating preneoplasia by increased saturation density followed by progression to transformation. These parameters are driven by Darwinian selection of spontaneously occurring, cumulative mutations, in accordance with recent genomic analyses of human cancer, just as it is in the evolution of species. The cell culture model will allow correlation of the preneoplastic increases in saturation density with genetic changes, and development of methods for demarcating fields during surgery so that they can be excised along with the tumor, thereby reducing the possibility of recurrence at the site.

Saturation density of skin fibroblasts as a quantitative screen for human cancer susceptibility.

Genomic analysis of human cancers reveals a large number of genetic changes per cell that presumably underlie development of the disease. The complexity of these changes that differ from one type of cancer to the other and from patient to patient with the same type of cancer raises questions about the feasibility of genomic analysis as an indicator of susceptibility to cancer. However, skin fibroblasts (SF) from individuals in families with heritable forms of cancer, and from cancer-bearing individuals, show correlation with a significant increase in saturation density (SD), as well as other neoplasia-related properties. Procedures are described for amplifying and quantifying differences in SD on the basis of studies of spontaneous transformation in the NIH 3T3 line of mouse fibroblasts. It is proposed that such procedures be evaluated as quantitative screens for susceptibility to cancer in the general population.

Rethinking "cancer as a dynamic developmental disorder" a quarter century later.

In a Perspectives article of 1985 in Cancer Research, I discussed evidence for a nonmutational origin of teratocarcinoma, and cited related claims in liver carcinogenesis, which I later broadened to include spontaneous transformation in cell culture. Further research on spontaneous transformation cast doubt on that interpretation, and motivated re-examination of the cases for teratocarcinomas and liver cancer. This re-examination led to the conclusion that mutation and selection underlie the development of neoplasia in all three cases. At the same time, it affirmed the primary role of hierarchical tissue structure and intercellular relations in maintaining homeostasis, which was the major theme of the 1985 article. Tumor promotion disrupts intercellular relations to allow progressively selective proliferation of initiated cells and accompanying clonal accumulation of genetic change.

Contact interactions between cells that suppress neoplastic development: can they also explain metastatic dormancy?

A comprehensive listing with accompanying discussions is given for established cases of interactions between normal and neoplastic cells of the same histotype that suppress neoplastic development. General principles that apply to the process are: (a) the requirement for a large excess of normal cells in direct contact with the neoplastic cells; (b) the effectiveness of suppression decreases with the degree of malignant progression of the neoplastic cells; and (c) the transformability of normal cells decreases under long-term negative selection, which also increases their contact suppression of neoplastic cells. Although suppression requires adhesive contact, it does not require gap junction communication, and it represents the first line of defense against tumor development. In contrast, potentially metastatic cells released from primary carcinomas into the circulation are activated to multiply when they form heterotypic adhesions with endothelial and parenchymal cells of distant organs. The great majority of the disseminated cancer cells (DCCs) fail to develop heterotypic cells adhesions in distant organs, and remain metastatically dormant as single cells. The lack of growth factors for the DCC in foreign territory also contributes to metastatic dormancy. The major insights about suppression of neoplastic development by homotypic contact arose from strictly operational experiments with living cells in culture. Molecular characterization of the cell-cell adhesions that underlie neoplastic suppression and metastasis activation has had only limited success, probably because of the complex variety of molecules involved. Hence, a program is outlined for further operational experiments on cell-cell interactions in tumor suppression to deepen our understanding of the neoplastic process, and provide possible avenues for its control.

Cell-cell contact interactions conditionally determine suppression and selection of the neoplastic phenotype.

Separation of chemical and physical carcinogenesis into the stages of initiation (mutation) and promotion (selection) established that incipient neoplastic cells could persist in the organism indefinitely without expression. Spontaneous mutations associated with cancer also lie dormant in untreated normal tissue. Without selection, there is no tumor development. Experiments in cell culture showed that confluent normal fibroblasts suppress growth of contacting transformed fibroblasts, and that normal keratinocytes similarly suppress tumor formation by adjacent papilloma cells. With cells that are generally more susceptible to transformation, however, prolonged contact inhibition progressively selects mutants that favor neoplastic growth. Selection of individual mutant cells allows them to become a significant fraction of the population and creates an enlarged target for additional genetic hits. Crucially, this enrichment step, not the initial mutation step, is the numerically limiting factor in tumor development. Unexpectedly, variants that are resistant to spontaneous transformation are selected in vitro by growing cells for many low density passages at maximal exponential rate. Confluent cultures of resistant variants suppress the growth and normalize the morphology of contacting transformed cells. Varying the conditions for selection shows that tumorigenic transformation is preceded by intermediate steps of progressively higher saturation density that are increasingly permissive for the expression of the more neoplastic cells in the population. There is also evidence of increasing permissiveness with age of normal tissues in vivo for solitary cancer cells transplanted in their midst. Spontaneous transformation in culture can be used to identify dietary components that are required for promotion and may therefore be applicable in prevention of human cancer.

Ordered heterogeneity and its decline in cancer and aging.

Ordered heterogeneity was introduced as a basic feature of the living state in the mid-1950s. It was later expanded to "order in the large over heterogeneity in the small" as the first principle of a theory of organisms. Several examples of ordered heterogeneity were given at the time to illustrate the principle, but many more have become apparent since then to confirm its generality. They include minimum size requirements for progressive embryological development, the errant behavior of cells liberated from tissue architecture, their sorting out to reconstitute tissues on reaggregation, and contact regulation of cell proliferation. There is increasing heterogeneity of cell growth with age, and marked heterogeneity of many characters among cells of solid epithelial tumors. Normal growth behavior is reintroduced in solitary, carcinogen-initiated epidermal cells by contact with an excess of normal epidermal cells. Contact normalization also occurs when solitary hepatocarcinoma cells are transplanted into the parenchyma of normal liver of young, but not of old, animals. The role of the plasma membrane and adhesion molecules in ordering heterogeneity is evaluated. Organizing the results in a conceptual structure helps to understand classical observations of tumor biology such as the lifetime quiescence of carcinogen-initiated epidermal cells and the marked increase of cancer incidence with age. The principle of order above heterogeneity thus provides a unifying framework for a variety of seemingly unrelated processes in normal and neoplastic development. Whereas contact between cells is required for these processes to occur, gap junctional communication is not required.

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.

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".

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.

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.

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.

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.