Automatic enumeration and characterization of heterogeneous clonal progression in cell transformation.

Most human tumors are clonal in origin, although the cells may be diverse in their properties. Since the tumors evolve through progressive stages over decades of time, it is possible that the conditions that induced the tumor transform many cells, but that selective overgrowth of the fastest growing lead to a clonal population of identified tumor cells. We studied the progression of neoplastic transformation in clones from a population in which about 10% of the cells formed well-defined transformed foci. A few of the clones produced many large foci, but most of the clones produced no foci or only one focus. Maintenance of the nonproducing and low-producing clones under the growth constraint of confluence and low serum concentration, which promotes transformation, led to the production of large numbers of small foci by all of them. Visual inspection revealed considerable heterogeneity in size and density among the foci from each clone, and this was quantitated by computer scanning. Subclonal analysis of focus formation was done on one of the clones after it had undergone further growth constraint to promote transformation. As in the original cloning, some of the subclones produced many large foci, but most produced none. Another round of growth constraint was imposed on the nonproducing subclones, which then became producers of many small foci varying in size and density. The results indicate that most if not all cells in the population respond to growth constraint by undergoing transformation. Though there is wide variation in the degree of transformation, the results are consistent with the view that an entire field of cells exposed to carcinogenic conditions in an animal undergoes some progression toward neoplasia but that heterogeneity of the response followed by selective neoplastic growth may lead to a clonal origin of the clinically detected tumor.     

Evidence for the progressive and adaptive nature of spontaneous transformation in the NIH 3T3 cell line.

The NIH 3T3 mouse cell line is widely used as a recipient of DNA from tumors to demonstrate the presence of transforming oncogenes. We show that these cells produce transformed foci spontaneously if kept in the confluent state for more than 10 days. The formation of foci depends on the type and concentration of bovine serum used in the medium and passage history of the cells. Cells maintained in continuous exponential multiplication in the subconfluent state by transfer every 2-3 days in medium with 10% calf serum failed to develop the capacity to produce foci in 2% calf serum, but those transferred the same way in 2% calf serum or in 10% fetal bovine serum, which is a less potent growth stimulant, did develop that capacity to an increasing degree over time. The number of transformed cells increased sharply with the time that a culture remained in the confluent state. There are several morphological types and degrees of transformation, which indicates that the underlying changes are varied and the process is progressive. The results also suggest that transformation occurs in a fraction of an entire cell population that is undergoing a physiological adaptation to moderate constraints on its growth.     

Mutual interactions between telomere heterogeneity and cell culture growth dynamics shape stochasticity of cell aging

Mathematical modeling and computational simulations are often used to explain the stochastic nature of cell aging. The models published thus far are based on the molecular mechanisms of telomere biology and how they dictate the dynamics of cell culture proliferation. However, the influence of cell growth conditions on telomere dynamics has been widely overlooked. These conditions include interactions with surrounding cells through contact inhibition, gradual accumulation of non-dividing cells, culture propagation and other cell culture maintenance factors. In order to follow the intrinsic growth dynamics of normal human fibroblasts we employed the fluorescent dye DiI and FACS analysis which can distinguish cells that undergo different numbers of divisions within culture. We observed rapid generation of cell subpopulations undergoing from 0 to 9 divisions within growing cultures at each passage analyzed. These large differences in number of divisions among individual cells guarantee a strong impact on generation of telomere length heterogeneity in normal cell cultures and suggest that culture conditions should be included in future modeling of cell senescence.     

Genetic heterogeneity in isogenic homozygous clonal zebrafish.

The C32 isogenic homozygous diploid (IHD) strain of the zebrafish (Danio rerio) was found to be polyallelic at a malate dehydrogenase locus (sMdh-A). A variant allele is thought to have arisen via mutation within the past 10 bisexual generations that have maintained the strain since its last gynogenetic cloning event; this unique allele now predominates at the sMdh-A locus. The estimated mutation rate in this species is sufficiently high that long-term genetic homogeneity of its IHD clones cannot be assumed. Researchers using such bisexually maintained clones should be aware that they are not necessarily using genetically uniform subjects. Genetic uniformity of cloned IHD zebrafish will be maximized if experimental subjects are obtained soon after a cloning event.     

Relation between growth hormone and Cortisol spontaneous secretion in children

OBJECTIVE Dual effects of corticoids upon GH release in man have been described: a rapid, potentiating effect and a delayed suppressive one. Many investigators have used pharmacological doses of glucocorticoids in an attempt to detect this interaction of corticoids and GH. The aim of the present study was to determine the relationship between GH and Cortisol spontaneous secretion in children under physiological conditions.
DESIGN AND PATIENTS Nine children aged 7.5–13.5 years with different growth rates were evaluated over a 24-hour period. Four were of normal stature (±2 SD) and five were below 2 SO (familial or constitutional short stature). Blood samples were obtained at 20-minute intervals over a 24-hour period.
MEASUREMENTS GH and Cortisol concentrations were measured by radioimmunoassay in each of the 73 samples collected.
RESULTS A significant correlation was obtained between mean 24-hour Cortisol concentration and mean 24-hour GH concentration ( r = 0.83; P < 0.01), the sum of 24-hour GH pulse amplitudes ( r = 0.85; P < 0.01) and the highest 24-hour GH concentration ( r = 0.88; P < 0.01). A significant positive correlation was also detected between mean 24-hour Cortisol concentration and the number of GH pulses over a 24-hour period ( r = 0.70; P < 0.05). However, there was no significant correlation between mean 24-hour GH concentration and number of Cortisol pulses over a 24-hour period. The correlation between number of Cortisol pulses and number of GH pulses was also non-significant.
CONCLUSION These data suggest an interplay of spontaneous GH and Cortisol secretion in children. Physiological plasma Cortisol levels may play a role in brain-pituitary regulatory mechanisms of GH secretion.     

Phenotypic transformation of clonal myogenic cells to cells resembling chondrocytes.

The nicotinamide analogue 6-aminonicotinamide and dibutyryl 3':5'-cAMP inhibit myogenesis in a clonal rat cell line from skeletal muscle. Both reagents produce a similar morphological response in the cells, and stimulate collagen and glycosaminoglycan synthesis. These data suggest that 6-aminonicotinamide and dibutyryl cAMP induce a phenotypic transformation of myogenic cells to cells that share many characteristics with chondrocytes.     

Adaptive evolution of degrees and kinds of neoplastic transformation in cell culture.

Human cancers undergo protracted complex development from benign to malignant states, as most thoroughly documented in the mole-to-melanoma sequence. The early stages of the sequence tend to redifferentiate into normal tissues; the later stages progress to ever increasing multiplication and malignancy. When placed under the growth constraint of either crowding or low serum concentrations, the NIH 3T3 line of mouse cells readily undergoes transformation, expressed in the development of foci of cells that continue to multiply at confluence when the rest of the population has stopped. If the nontransformed cells are maintained for 3 months by frequent low-density passages in high concentrations of calf serum, they gradually lose the capacity to undergo transformation when the constraints are applied. The same conditions of passage have been used to reverse the transformation, both processes resembling in principle the reversal of the early stages of the mole-to-melanoma sequence. When the frequent low-density passages are made in high concentrations of fetal bovine serum, which supports a slightly lower growth rate than an equal concentration of calf serum, the degree of transformation is gradually increased, so that the foci become more numerous, broader, and thicker, reaching a maximum in successive assays at about 3 months of passaging. A diversity of focal morphologies is sporadically generated in the calf serum passage by exposing the cells to various concentrations of calf serum for 14 days of growth and confluency before assaying them. The dependence of the number, density, and morphology of foci on the environment in which the cells had been grown before assay reinforces the evidence that the transformation is an epigenetic process. The fact that these effects develop in culture gradually over an extended period of time suggests parallels to the characteristically long-term early regression and later progression, as well as the diversity of the mole-to-melanoma sequence, and may also be representative of other cancers.     

Clinical syndromes of transformation in clonal hematologic disorders.

The majority of clonal hematologic syndromes, including lymphoproliferative, myeloproliferative, and myelodysplastic disorders, tend to undergo transformation. However, the frequency of transformation varies widely. For example, transformation is almost invariable in chronic myelogenous leukemia, but it is infrequent in other myeloproliferative disorders. Similarly, transformation occurs in approximately 33% of follicular lymphomas but less commonly in other lower-grade lymphomas. At a genetic level, although some secondary lesions are seen across the spectrum of transformation syndromes (such as loss of function of p53 and p15/p16), there is considerable intra- and interdisease variability, with no common denominator. This review of the literature will discuss these transformations, noting their frequency, pathologic changes observed, clinical syndromes described, underlying genetic correlates, and prognostic and therapeutic implications.     

Crucial role of thyroid hormone in x-ray-induced neoplastic transformation in cell culture.

Incubation of mouse embryo fibroblasts (C3H/10T1/2) in media depleted of thyroid hormone for 1 week rendered the cells completely resistant to the transforming action of an x-ray dose, 4 grays, that yields transformation frequencies (no. foci per surviving cells) of approximately 10(-3) in media supplemented with triiodothyronine (T3) (1 nM). Studies on the timing of the additions or removal of the hormone indicate that T3 was maximally effective when added 12 hr before irradiation and that progression from the time of irradiation to the appearance of foci (6 weeks) was independent of the presence or absence of the hormone. The dependence of x-ray-induced transformation on the concentration of T3 in the medium was virtually the same as that for augmentation of Na+,K+-ATPase activity. The latter effect was used as a measure of T3 induction of protein synthesis. A further indication of the involvement of protein synthesis in the process is the abolition of T3- and x-ray-dependent transformation by cycloheximide at a concentration (100 ng/ml) that inhibits 50% of protein synthesis. We propose that thyroid hormone induces the synthesis of a host protein that is an obligatory participant in x-ray-mediated transformation.     

Nerve growth factor-mediated induction of tyrosine hydroxylase in a clonal pheochromocytoma cell line.

We have established a clonal cell line, PC-G2, from an experimentally induced rat pheochromocytoma. Administration of nerve growth factor to PC-G2 causes a 4- to 8-fold induction in the specific activity of tyrosine hydroxylase [tyrosine 3-monooxygenase; L-tyrosine,tetrahydropteridine:oxygen oxidoreductase(3-hydroxylating); EC 1.14.16.2]. The response is elicited in a dose-dependent fashion, at concentrations above 0.1 microgram/ml. Antiserum to nerve growth factor inhibited the induction of tyrosine hydroxylase. Dexamethasone enhances the nerve growth factor-mediated elevation of tyrosine hydroxylase. After 3--4 days of exposure to nerve growth factor the maximal induction of tyrosine hydroxylase is seen, although a significant increase can be observed after 24 hr. In contrast to the PC-12 cell line (derived from the same tumor), in which neurite outgrowth occurs in response to nerve growth factor, there is no morphological change or alteration in growth rate of PC-G2 cells after exposure to nerve growth factor.     

Lentivector-mediated clonal tracking reveals intrinsic heterogeneity in the human hematopoietic stem cell compartment and culture-induced stem cell impairment

Knowledge of the composition and interrelationship of the various hematopoietic stem cells (HSCs) that comprise the human HSC pool and the consequence of culture on each class is required for effective therapies based on stem cells. Clonal tracking of retrovirally transduced HSCs in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice revealed heterogeneity in the repopulation capacity of SCID-repopulating cells (SRCs). However, it is impossible to establish whether HSC heterogeneity is intrinsic or whether the culture conditions required for retroviral transduction induce qualitative and quantitative alterations to SRCs. Here, we report establishment of a clonal tracking method that uses lentivectors to transduce HSCs with minimal manipulation during overnight culture without cytokine stimulation. By serial bone marrow (BM) sampling of mice receiving transplants, short-term SRCs (ST-SRCs) and long-term SRCs (LT-SRCs) were identified on the basis of repopulation dynamics demonstrating that their existence is not an experimental artifact but reflects the state of the HSC pool. However, 4 days of culture in conditions previously used for SRC retroviral transduction significantly reduced SRC number as assessed by clonal analysis. These studies provide a foundation to understand the molecular and cellular determinants of human HSC development and to develop therapies targeted to specific HSC classes.     

Glucocorticoid receptors and inhibition of bone cell growth in primary culture.

We have previously identified glucocorticoid binding proteins in cytosol of cells dispersed from fetal rat calvaria by collagenase digestion. The present study, employing primary culture of these cells, provides further evidence that these binding proteins represent glucocorticoid receptors. [3H]Dexamethasone bound to cytoplasmic extracts of cultured cells with an apparent Kdiss of 6.8 nM and exhibited approximately 8500 binding sites/cell. Nuclear translocation of [3H]dexamethasone was demonstrated with approximately 50% of bound steroid extractable from the nuclear pellet after incubation at 37 C; little nuclear transfer occurred at 0 C. The specificity of these binding site was characterized by competition studies with other steroids in whole cells, the order of affinities being: triamcinolone acetonide greater than dexamethasone greater than progesterone greater than cortisol greater than corticosterone = cortexolone. Non-glucocorticoids except progesterone competed only poorly. Sedimentation analysis of [3H]dexamethasone-protein complexes on sucrose gradients revealed a cytoplasmic peak of 6.5 S in salt-free gradients and 3.8 S in 0.3 M KCl gradients. Dexamethasone addition to the culture medium resulted in a dose-dependent inhibition of cell growth with approximately 40% reduction in cell number at 13 nM. That this inhibition was receptor mediated was substantiated by the partial blockade of the dexamethasone effect in the presence 1.3 microM progesterone. Functionally, dexamethasone inhibits the growth of these cells. These data provide evidence for receptor mediated inhibitory effects of glucocorticoids directly at the level of the bone cell.     

Contribution of phenotypic heterogeneity to adaptive antibiotic resistance

Antibiotic-resistant isolates of Salmonella enterica were selected on plates containing lethal concentrations of rifampicin, kanamycin, and nalidixic acid. The stability of the resistance phenotype was scored after nonselective growth. Rifampicin-resistant (Rif^r) isolates were stable, suggesting that they had arisen by mutation. Mutations in the rpoB gene were detected indeed in Rif^r mutants. In contrast, a fraction of kanamycin-resistant (Km^r) and nalidixic acid-resistant (Nal^r) isolates showed reduced resistance after nonselective growth, suggesting that mechanisms other than mutation had contributed to bacterial survival upon lethal selection. Single-cell analysis revealed heterogeneity in expression of the porin gene ompC, and subpopulation separation provided evidence that reduced ompC expression confers adaptive resistance to kanamycin. In the case of Nal^r isolates, mutations in the gyrA gene were present in most nalidixic acid-resistant isolates. However, the efflux pump inhibitor Phe-Arg-β-naphtylamide (PAβN) reduced the level of resistance in Nal^r mutants, indicating that active efflux contributes to the overall level of nalidixic acid resistance. Heterogeneous efflux pump activity was detected in single cells and colonies, and a correlation between high efflux and increased resistance to nalidixic acid was found. These observations suggest that fluctuations in the expression and the activity of critical functions of the bacterial cell, alone or combined with mutations, can contribute to adaptive resistance to antibiotics.Except for bacterial species that undergo developmental programs, bacteria have been traditionally viewed as clonal populations of identical cells. In fact, classic genetics and physiology have routinely used batch cultures of bacteria and individual bacterial colonies, assuming that all cells were identical. Because bacterial mutation rates are in the range of 10⁻¹⁰ per base pair per replication (1), most cells in a liquid culture or within a colony are isogenic indeed. However, genetic identity does not necessarily imply phenotypic identity. The existence of spatial organization in Escherichia coli colonies has been known for almost a century (2–4), and the occurrence of diverse gene-expression patterns inside a colony was described 25 years ago (5). These historic examples are not rare exceptions: in the last few decades, single-cell analysis has provided examples of phenotypic variability in bacterial populations made of isogenic cells, both under laboratory conditions and in natural environments (6–12).Phenotypic heterogeneity can be the consequence of chemical communication, leading to a heterogeneous response at the single-cell level (13). In other cases, however, phenotypic heterogeneity arises either as a programmed epigenetic event or at random, without the involvement of environmental cues. Classic phenomena involving programmed heterogeneity are the bifurcation of a bacterial population into two distinct states or “bistability” (14) and the reversible switching of gene expression or “phase variation” (15). Randomly generated heterogeneity is usually the consequence of noisy gene expression (16, 17). The distinction between programmed and random heterogeneity, albeit useful in practice, is not always clear-cut: quantitative differences caused by noise can become qualitative above a threshold, triggering a programmed response (18). Another source of heterogeneity is gene amplification, which spontaneously occurs in a fraction of cells within a bacterial population (19).The selective value of phenotypic heterogeneity can be envisioned in certain cases (6, 20). Furthermore, theoretical analysis supports the view that randomly generated phenotypic diversity can increase the chances of survival when bacterial populations are subjected to rapid, severe, or complex environmental fluctuations (21, 22). Such bet hedging strategies imply group selection, which has been considered intrinsically weak in classic population biology studies (23). However, models based on game theory suggest that strategies that generate phenotypic heterogeneity can provide an evolutionary advantage, despite the fact that they lower the immediate fitness of individual organisms (24–26). Models based on information theory also support the view that bet hedging can be advantageous in harsh and changing environments (27).Coevolution of bacteria with natural antibacterial compounds has fostered the evolution of resistance mechanisms, usually classified into three types: innate resistance, acquired resistance (e.g., by mutation and by horizontal transfer of genetic determinants), and adaptive resistance (28–30). Adaptive resistance typically involves environmentally induced gene-expression changes that increase the ability of a bacterium to survive in the presence of an antibiotic (31–36). In this study, we provide evidence that cell-to-cell fluctuations in the expression and activity of critical cellular functions can induce adaptive resistance to antibiotics in the absence of known environmental stimuli. Physiological differences preadapt certain cells within an isogenic culture to survive lethal selection. If a feedback loop propagates the physiological state that permits survival, growth of the “lucky” survivors generates a bacterial population with increased antibiotic resistance.     

Progenitor cells divide symmetrically to generate new colony-forming cells and clonal heterogeneity

Self-renewal is the most fundamental property of haemopoietic stem and progenitor cells. However, because of the need to produce differentiated cells, not all cell divisions involve self-renewal. We have used a colony replating assay to follow the fates of individual haemopoietic progenitor cell clones. For this, human myeloid colony-forming cells (CFCs) were cultured by standard methodology. Onset of proliferation and growth rates were established by a video recording method. Individual colonies were replated several times to document the rate of clonal extinction, and the numbers of secondary, tertiary and quaternary CFCs. The clonogenic population exhibited similar kinetics in terms of onset of proliferation and growth rate. Clonal extinction was progressive so that only 30 +/- 7% (mean +/- standard error of the mean; n = 4) of the original primary colonies formed quaternary colonies after the third replating step. However, individual primary CFCs that produced colonies throughout the experiment generated, on average, 40 +/- 8 secondary and tertiary CFCs overall. The values obtained in standard culture conditions were modified when granulocyte colony-stimulating factor (G-CSF) or G-CSF plus interleukin 3 were used to stimulate colony growth, showing that the kinetics of colony formation respond to extrinsic regulation. Examination of the replating potential of individual secondary colonies in the clones demonstrated that they generated different numbers of tertiary colonies. The data best fit a stochastic model of haemopoietic cell development where event probabilities can be modified by extracellular factors.     

Control of ovarian cell growth in culture by serum and pituitary factors.

An ovarian cell line was developed that requires hormonal conditioning of the host for growth in vivo and that requires special factors for growth in vitro. It is necessary to prepare special culture media to demonstrate the effects of growth factors in vitro. To this end, methods were developed for removing from serum those essential factors required for the growth of ovarian cells in culture. Minimal growth occurred in medium containing fetal calf serum that had been passed through a porcelain filter. This method of depleting serum was replaced by a procedure involving passage through a carboxymethylcellulose column. Either pituitary extract or the eluate from the column restored growth in these depleted media. The eluate was more active than pituitary extract with regard to maximal growth enhancement. When the cells were incubated in the depleted media, viability, as measured by plating efficiency, decreased with incubation time. Either pituitary extract or the eluate from the column prevented such death of cells. Based on these findings, we postulate that the eluate contains both a survival factor and a growth-promoting factor for these ovarian cells, while pituitary extract contains only the survival factor.     

Evolution of tumor cell heterogeneity during progressive growth of individual lung metastases.

The metastatic properties of tumor cell clones isolated from individual lesions of B16 melanoma metastatic to lung have been examined at different stages in the evolution of metastasis. Clonal analysis of metastatic lesions produced by B16 melanoma populations containing clones with identifiable, stable drug-resistance markers revealed that the majority (greater than 80%) of experimental metastases produced by intravenous injection of tumor cells are of unicellular origin. During the early stages of their growth (less than 25 days after initial tumor cell arrest), the majority of metastatic lesions contain cells with indistinguishable metastatic phenotypes (intralesional clonal homogeneity) although different clonally homogeneous lesions from the same host contain tumor cells with different metastatic phenotypes (interlesional clonal heterogeneity). Progressive growth of metastatic lesions is accompanied by emergence, within originally clonally homogeneous lesions, of variant tumor cells with altered metastatic properties (intralesional clonal heterogeneity). By 40-45 days after initial arrest of injected tumor cells in the lung, 90% of the metastatic lesions are populated by cells with heterogeneous metastatic phenotypes.