BACKGROUND Clinical and experimental observations indicate that resistance to anticancer drugs may be spontaneously reversible over time. MATERIALS AND METHODS This work is a mathematical and statistical analysis of the relationship, during a 9-month experiment, between the resistance of repeatedly re-seeded hepatoma cells to methotrexate (MTX) or to cisplatin (cisP) and untreated cell proliferation, telomere length and telomerase activity. RESULTS All variables showed complex oscillations, as previously published. In this work, cell proliferation was modelized by the logistic model, and the proliferation rates (a-values) together with their variations (va-values) were calculated. CONCLUSION Significant correlations were discovered between cell resistance to treatments and a-values, va-values, telomere length and telomerase activities. These results open new insights into the handling of chemotherapy in the treatment of cancers.

The proliferation rate of various cell types in vitro, including hepatoma Fao cells, displays aperiodic oscillations. The frequency of these oscillations is about one every 3-5 weeks, and there are variations in cell functions and polarity. Topological analysis has showed that these oscillations in growth rate are determined, and presumably chaotic. One characteristic of complex chaotic systems is that their dynamics can be persistently modified by a small external perturbation. We show that treatment with a single small dose of the anticancer drug methotrexate causes long-term stable alteration of the oscillatory dynamics of Fao cell proliferation. The oscillations of growth rate are shifted, and their mean level decreased according to a fractal pattern. (c) 2005 Elsevier SAS. All rights reserved.

Clinical and experimental observations indicate that resistance to anticancer drugs may be spontaneously reversible over time, but the mechanisms of this reversal are unknown. The resistance of cultured hepatoma cells to methotrexate (MTX) and cisplatin was followed for 9 months. Cells were exposed to three treatments: MTX 200 nM for 24 h or 15 nM continuously and cisplatin 50 microM for 2 h. We investigated the relation between the temporal pattern of cell resistance and the previously reported fluctuations in cell proliferation rate, telomere length and telomerase activity. Spontaneous major peaks in resistance to each drug fell in time windows of 2-3 months (60-70 population doublings) and were at different times for each drug. The frequency of the fluctuations in drug resistance was the same as that of variations in cell growth rate, but amplitudes were unrelated. By contrast, resistance was directly related to telomere length dynamics in the same cells. MTX resistance occurred when telomeres shortened and cisplatin resistance when they were elongated. Furthermore, peaks of resistance to the continuous treatment with MTX were observed at 350-bp intervals of mean telomere length (9.06, 9.41, and 9.76 kbp) during the two 2-month phases of telomere shortening. Statistical analysis demonstrates the sinusoidal relationship between intermittent MTX resistance and telomere length. Possibly, erosion of telomeres encroaches on periodically spaced nucleosomal proteins, defining the onset of resistance phases. This evidence that resistance of tumoral cells to anticancer drugs may be intermittent and that onset of resistance is dictated by telomere length has major implications for clinical practice.

OBJECTIVE:The production of blood cells in vivo, both normal and tumoral, displays oscillatory dynamics. Many cells in long-term cultures also show large amplitude oscillations of proliferative rate. Therefore we examined the proliferation dynamics of mouse bone marrow cells (MBM) and their clonogenic progenitor production (BMP), in order to characterize these dynamics. METHODS:Five Dexter-type cultures of MBM cells and their clonogenic BMP production were examined for up to seven-months periods of time. The recorded time series exhibited a complex pattern of oscillations with variable amplitudes. We previously reported a method that allowed analysis of such nonlinear dynamics of hepatoma cell proliferation. We applied this method, based on the two-dimensional recurrent representation of data, to analyze the fluctuations of bone marrow cells proliferation. RESULTS:The proliferation rate of mouse bone marrow cells shows large amplitude oscillations every 2 to 3 weeks. Mathematical analysis revealed a deterministic mechanism that controls all proliferation local maxima of MBM cells. Dynamics for progenitor production resembled that of parental cells. This reflects a predominant negative feedback on bone marrow cell proliferation. CONCLUSION:These dynamics were opposite of that previously described for hepatoma cells where the dominant control is applied to the local minima (troughs of proliferation). Therefore, the complex system of cell proliferation is controlled by a bipolar mechanism, with a predominant dampening command depending on the cell type. We propose that the dominant dampening control of local maxima in bone marrow cells protects the stock of stem cells.

Non-linear dynamics, often chaotic, are now accepted as the normal way in which major physiological functions proceed, allowing adaptation. The length of telomeres, the chromosome endings, is critical in limiting cell lifespan and in controlling subsets of downstream genes. Telomere length dynamics in tumoral cells is the net result of mitotic erosion of telomeric DNA and telomere repair by the enzyme telomerase. We observed telomere length oscillations in the long-lived hepatoma Fao cell line, for forty three 6-day passages in culture. Telomerase activity oscillated with similar frequency and opposite amplitude. We mapped the combined data of telomere erosion and telomerase activity. There was reverse symmetry between consecutive periods of 10 passages. Thus, telomere length and telomerase activity can be considered to be a single complex system with strong reciprocal regulation.

The proliferative activity of long-term cultured mammalian cells exhibits traits of a complex dynamic system, with a succession of spontaneous rises and falls in proliferation rate. We analyzed three successive series of proliferation data for the Fao hepatoma cell line in longterm cultures. In the three series the proliferation rate displayed apparently disordered oscillations, which each lasted about 3-5 passages, with variable amplitude and were therefore unpredictable. Such non-linear kinetics raises the major issue of whether these fluctuations are random, or determined and coordinated. We used a graphical method of analysis of the data, which demonstrated that all troughs of proliferation were mathematically related to a common value in each series. This common value was itself related to the maximum level of proliferation of the cell line. Non-linear analysis thus confirmed that the fluctuations in proliferation rate of tumoral Fao cells are, at least in part, determined. This pattern evokes chaotic dynamics and is evidence for the flexible coordination of the complex system linking positive and negative growth regulators in long-term cultured cells.

Immortal cells perpetuate the rises and falls of proliferation that are progressively damped in mortal long-term cultured cells. For immortal rat hepatoma Fao cells, similar waves of proliferation occurred about every 3–4 wk. Under the same conditions, embryonic human fibroblasts and transformed but not immortalized embryonic fibroblasts display similarly recurring proliferation waves that progressively decrease in amplitude until senescence of the lines. In addition, strains of diploid normal human skin fibroblasts cultured under different culture conditions display a similar time-pattern of proliferation. Although the amplitude and baseline of these fluctuations are characteristic for each cell line, a common point was marked slow down in proliferation after every sequence of about 25 population doublings for all cells. Renewed proliferation waves of Fao cells allow about 22–23 additional population doublings each. Normal embryonic fibroblasts culture and its transformed counterpart accumulate about 30 and 60 population doublings, respectively, before senescence. Normal fibroblast strains accumulate about 25 population doublings over their entire life spans. This halt in proliferation after every stretch of about 25 population doublings may correspond to a structural or functional stop following attrition of telomeric DNA. This putative stop may be bypassed once in transformed embryonic cells and repetitively in immortal cells. In support of this hypothesis, we observed rapid telomere shortening, in two steps, during divisions of mortal embryonic cells, and maintenance of long telomeres in immortal Fao cells, which may indicate episodic repair of telomeres. Alternatively, such maintenance of long telomeres may reflect survival and successive clonal growth of rare cells with long telomeres. We suggest that the balance between telomere attribution and repair processes regulates the waves of proliferation.

This study focused on the activation/differentiation of human microglial cells and astrocytes, which is a prerequisite for HIV1 replication. In vitro, IFN gamma induced a differentiation-like morphological change in embryonic microglia and astrocytes, in both primary and in purified culture. This effect was enhanced by TNF alpha which in itself had no effect. IFN gamma did not increase the low level of endogenous TNF alpha, which is not secreted by embryonic cells, but stimulated the expression of TNF alpha R1, although to a relatively low extent. IFN gamma facilitated TNF alpha response through an increase in TNF alpha R1 expression, but probably also through interaction with different intracellular signal transduction pathways.

A human fibroblastic cell line transformed by the SV40-T antigen sequence and continuously cultured for 7 months displayed large periodic variations in cell proliferation. This contrasted with other characteristics of this cell line that remained constant: mosaic cell shape, absence of cell contact inhibition, and predominance of a hypodiploid population. Similar fluctuations in proliferative capacity were also found during the long-term growth of a transformed but nonimmortalized human fibroblastic line prior to senescence, and in the established hamster fibroblastic Nil cell line. This growth pattern suggests a recurrent stimulation of growth in these three transformed cell lines. The proliferation pattern from cultured transformed cells may thus be complex and requires further investigation, These variations presumably influence major cell functions. This observation has important implications for the analysis of data from such cell lines.

The incorporation of radioactivity from [1-14C]galactose into TCA-precipitable material was determined in skin fibroblasts derived from 11 galactosemic patients deficient in galactose 1-phosphate uridyl transferase (GALT-). "R" ratios (designated the R phenotype) were defined as the ratio between [14C]galactose incorporation and [3H]leucine incorporation. Results were expressed as a percentage of the controls. In the GALT-strains this ratio varied from strain to strain, presumably depending on the efficiency of the secondary route via the UDP-galactose pyrophosphorylase pathway. In 10 GALT-patients without late serious clinical manifestations, the R phenotype varied hom 37 to 57% of the control value. In the 11th patient, the R phenotype was only 20% of the control. Thus, we obtained a significantly lower R phenotype in one patient who was distinguished from the others by having very severe delayed neurological complications, although compliance to galactose-free diet was good. We suggest that, in this patient, the development of the UDP-galactose pyrophosphorylase pathway was not sufficient to ensure the availability of enough galactose for the necessary synthesis of glycoproteins and glycolipids. Thus the R phenotype may be an indicator of the risk of late neurological complications. The determination of the R phenotype of GALT-patients may therefore be valuable. However, further investigations of galactosemic patients with neurological complications are required to confirm this relationship.

Fructose strongly stimulates the growth of normal diploid human skin fibroblasts (SFs) and induces marked changes in their morphology and lipid accumulation. This mitogenic effect occurs despite very low fructose consumption and depends on the presence of glutamine. The cell kinetics of cultured fructose-fed human skin fibroblasts were different from those fed on glucose: in the presence of fructose a high proliferative index persisted at Day 14 of culture and the duration of the total cell cycle and of the G1+1/2 M and S phases was slightly shorter. The mitogenic effect of fructose on SF was largest in the presence of human serum: it was small or undetectable when fibroblasts were cultured in media supplemented with dialyzed human serum, fetal bovine serum, or serum substitutes. This suggests that serum growth factor(s) mediate the mitogenic effect of fructose. Only normal diploid human cells seem to be sensitive to this mitogenic effect of fructose: the long-term growth of normal human liver cells on fructose was slightly better or similar to that on glucose. In contrast, fructose could only support limited growth of hamster fibroblastic Nil cells and of a transformed human fibroblastic line, which grew better with glucose.

In skin fibroblasts of patients presenting with galactosaemia, either from galactose 1-phosphate uridyltransferase or galactokinase deficiency, a deficit in extracellular glucose utilization was observed. This deficit was constant over 3 weeks of continuous cell growth in a medium containing 5.5 mmol/L glucose as the only hexose, and homologous serum. Levels of glucose utilization by deficient skin fibroblasts were stable at about 65-70% of the glucose utilization of control normal skin fibroblasts. Cell morphology was normal, and cell growth was subnormal during this period. However, the energy provision appeared sufficient for cellular needs since cell growth in this glucose medium was observed not to depend on the presence of extracellular glutamine. In contrast, glutamine was required for growth of galactosaemic fibroblasts cultured in medium containing 5.5 mmol/L galactose. If expressed in many cell types, this impaired glucose uptake would be expected seriously to damage highly glucose-dependent tissues such as the central nervous system. This might be of relevance to the persistent neurological damage observed in many galactosaemic patients in spite of their compliance with an early strict galactose-free diet.

The activity of Glutamine Synthetase (GS) was measured during the growth of human diploid skin fibroblasts cultured for three weeks in the presence or absence of either glucose or glutamine or both. In medium free of both glucose and glutamine, a single late peak in GS activity was observed concomitantly with delayed small cell protein increment. In all media containing either glucose or glutamine or both. GS activity rose sharply during rapid cell growth, displayed a plateau, and then decreased once the cells had reached confluency. The variations in extracellular amino acid levels were also determined and were found to depend on the composition of the medium but not on the cell culture duration. These results demonstrate, for the first time as far as we know, that strong GS activity is present in rapidly growing skin fibroblasts. In contrast to many other mammalian cell types, GS activity in human skin fibroblasts appears not to be subject to regulation by extracellular glutamine. This difference may well be connected with cell differentiation.

The combined effects of carbohydrates and glutamine were investigated in diploid strains of normal human skin fibroblasts cultured for 21 days under eight different culture conditions: hexose-free medium or medium containing D-glucose, D-galactose, or D-fructose, with or without added glutamine. Cell growth, hexose consumption, lactate production, intracellular glycogen content and extracellular amino acid levels were measured every third to fourth day. In the presence of glutamine, cells reached a higher saturation density in fructose medium than in glucose or galactose medium but per cell consumption of fructose and galactose was much less than that of glucose. Consumption of all three carbohydrates per unit cell growth exhibited three distinct phases: Days 1-3, 3-10, and 10-20, respectively. In the absence of glutamine the rate of cell growth was not altered in glucose or galactose medium, but slowed down considerably in fructose medium. Glutamine deprivation also led to changes in hexose consumption. In hexose-free media the cell growth rate at first was very slow, but rose after 2 or 3 weeks of culture. The levels of extracellular nonessential amino acids varied according to medium and growth phase. One of the most exciting findings was that human fibroblasts are able to maintain a slight excess of glutamine in all media not supplemented with glutamine and, more surprisingly, to synthesize it in a medium containing galactose and glutamine.

Human infant skin fibroblasts and liver cells were subcultured with 250 microM PUFA (polyunsaturated fatty acid), and primary cultures of glial brain cells from new-born rats with 100 microM; oleic acid was added to controls. Minimum essential medium (MEM) supplemented with bovine serum was used as a reference. During the short-term experiment (18-24 h), control liver cells showed a regular increase in protein level, while protein increment was more rapid in linoleic and especially in arachidonic acid-treated cells, but only for the first 3 hours. During the long-term experiment (7 d), control skin fibroblasts showed a faster growth rate (increase in number of cells) than reference or fibroblasts cultured with the added PUFAs. Lipid droplets were seen in the PUFA-treated liver cells and skin fibroblasts, and ultrastructural modifications were observed in fibroblasts, but without growth rate alteration. During the long-term experiment (2 w), control glial brain cells showed faster protein increment (measuring growth rate) than PUFA-treated cells, particularly than arachidonic acid-treated cells. HMGR (3-hydroxy-3-methylglutaryl-CoA reductase) activity, determined after 6 h (liver cells) or 1 and 2 w (brain cells) of culture, was low in controls and reference, whilst higher in PUFA-treated-cells, and was especially high in arachidonic acid-treated brain cells. The present study indicates than the high HMGR activity may correspond to cultures of cells rapidly stopped in their protein increment, and to cultures of cells showing a slow rate of proliferation. This contrasts with results obtained from in vivo experiments; it also emphasizes the high mevalonate (MVA) level as a possible sign of nutritional medium imbalance.(ABSTRACT TRUNCATED AT 250 WORDS)

SummaryThe localization of factor VIII procoagulant antigen (VIII: Ag) and factor VIII von Willebrand antigen (VWF: Ag) was investigated in human liver, lung, spleen, placenta and umbilical cord, by an immunoperoxidase technique using an avidin biotin complex (ABC). Positive staining for VIII: Ag was observed in the endothelial cells of liver sinusoids, veins and arteries, as well as in the endothelial cells of placenta, lung and spleen. VWF: Ag was detected in the vascular endothelial cells of all the organs explored. The staining intensity of both VIII: Ag and VWF: Ag varied in the different tissues and showed a distinctive pattern of distribution in the liver. VIII: Ag was also observed in the cytoplasm of dysplastic, foetal-like hepatocytes which infiltrated one liver specimen. Our results agree with the view that liver endothelial cells are a major site of Factor VIII (F VIII) storage and secondary release into the circulation. However, the bright staining intensity of VIII: Ag and VWF: Ag in the lung and placenta suggests that these two tissues might also be a substantial source of F VIII.

UNLABELLED Many publications indicate the beneficial effect of n-6 polyunsaturated fatty acids (n-6 PUFAs) in the control of coronary heart disease and diabetes, although the mechanism is not clear. Some of our previous results suggest that, in contrast to other lipids, n-6 PUFAs could have a permissive effect on carbohydrate oxidation. To check this hypothesis, we determined pyruvate dehydrogenase (PDH, decarboxylase: EC 1.2.4.1) activity in infant skin fibroblasts (ISF) incubated 6 hours in the presence of 0.25 mM linoleic (LI) or arachidonic (AR) acid, compared to oleic acid (OL) and control ISF incubated without addition of fatty acids. The four groups of cells were preincubated 36 hours either in the presence of fetal bovine serum (FBS), or in the presence of lipoprotein-deprived serum (LPDS). RESULTS (1) When the ISF were maintained in the medium containing FBS, the two PUFAs had little inhibitory effect on PDH activity, in contrast with the effect of OL. (2) When the ISF were kept in the lipoprotein-deficient medium, PDH activity was low in controls and in the OL cells, but the addition of LI or AR increased the activity. This suggests the role of n-6 PUFAs in enhancing carbohydrate oxidation, under certain conditions.