Neutropenia dynamics in a case of T-LGL lymphoproliferation illustrate rapid turnover of granulocyte progenitors.

OBJECTIVES: To elucidate the natural history of T-cell large granular lymphocyte (T-LGL) lymphoproliferation, we followed changes in associated fluctuating neutropenia for 3 years in an untreated patient presenting with the disease. MATERIALS AND METHODS: We report a nonlinear mathematical analysis of irregular neutrophil fluctuation, using iterative data maps, to detect long-term regulation of the neutrophil population. RESULTS: This geometric analysis indicated that variations of this sequence of neutrophil counts followed bounded deterministic dynamics around a fixed low level equilibrium, a situation similar to that previously observed for cultured mouse early bone marrow progenitor cells. CONCLUSION: These findings illustrate how the deleterious effect of T-LGL on neutrophils is balanced, over periods of years, by pulses of compensatory neutrophil production, potentially accounting for the commonly observed prolonged indolent course of the disease.

Prolonged perturbation of the oscillations of hepatoma Fao cell proliferation by a single small dose of methotrexate.

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.

Telomere dynamics determine episodes of anticancer drug resistance in rat hepatoma cells.

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.

Replicative senescence in normal liver, chronic hepatitis C, and hepatocellular carcinomas.

There is growing evidence that senescent cells accumulate in vivo and are associated with the aging process in parallel with the progressive erosion of telomeres. Because recent data show that telomere shortening is involved in the pathogenesis of liver cirrhosis, we looked for replicative senescence cells in normal livers, chronic hepatitis C, and hepatocellular carcinoma (HCC). Replicative senescent cells were detected on liver tissue cryosections using expression of a specific marker, senescence-associated beta-galactosidase, a cytoplasmic enzyme detected at pH 6. A total of 57 frozen liver samples (15 normal liver, 32 chronic hepatitis C, and 10 HCCs) were studied. Replicative senescence was graded as absent in 56% of cases (32 of 57) and present in 44% (25 of 57). Replicative senescence was considered present in 3 of 15 normal livers (20%), 16 of 32 chronic hepatitis cases (50%), and 6 of 10 HCCs (60%). In the group of nontumoral livers, the presence of senescent cells in liver was associated with older age (P =.03). In the group with chronic hepatitis C, fibrosis stage, but not activity grade, was significantly correlated with the accumulation of replicative senescent cells (P <.001). Finally, beta-Gal staining in nontumoral tissue was strongly correlated with the presence of HCC in the surrounding liver (P <.001). These results suggest that chronic hepatitis C represents a relevant model of accelerated replicative senescence and that accumulation of replicative senescent cells predispose to HCC development. Detection of replicative senescent cells may then serve as a predictive marker of a hepatocellular carcinoma in the surrounding tissue. HUM PATHOL 32:327-332.

Reversible induction of rat hepatoma cell polarity with bile acids.

A dynamic model for inducing and isolating polarized cell colonies from differentiated rat hepatoma was established with chenodeoxycholic acid (CDCA). Cells were treated with 75 microM CDCA in a 1% solvent mix (DMSO/ethanol: 0.5%/0.5%) for 11 days and positive Fao-BA1 and C2rev7-BA1 clones were isolated, respectively, from Fao and C2rev7. Cell polarization in these two clones was demonstrated by (i) the detection of (gamma)-glutamyl transpeptidase activity (gamma)-GT) and the presence of specific proteins, namely aminopeptidase N (APN), bile acid export pump (Bsep), multidrug resistance-associated protein 2 (Mrp2) at the canalicular pole, (ii) the expression of tight junction (ZO-1) and basolateral (1-18) marker proteins, (iii) the presence of regular microvilli in the cavities sealed by tight junctions, and (iv) functional bile canaliculi-like structures with the capacity to metabolise and secrete carboxyfluorescein diacetate dye. The polarized phenotype was maintained for more than 200 cell generations in the presence of CDCA and could be modulated by cell density or omitting the inducing agent. Hence this cellular model is well suited for studies on hepatic differentiation, polarization and bile salt trafficking with therapeutic implications.

Evidence for deterministic chaos in aperiodic oscillations of proliferative activity in long-term cultured Fao hepatoma cells.

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 long-term 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.

The time-pattern of rises and falls in proliferation fades with senescence of mortal lines and is perpetuated in immortal rat hepatoma Fao cell line.

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 fibroblast 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 attrition and repair processes regulates the waves of proliferation.

Evidence for interactions between rat hepatoma cell apoptosis and differentiation.

Partial copper depletion of a variant rat hepatoma cell line induces a transient inhibition of growth and the genesis of stable, well-differentiated revertants. We report a burst of cell death, synchronous with the peak of reversion. The characteristics of this cell mortality were typical of apoptosis and included detachment from the plastic support, chromatin condensation and fragmentation, and internucleosomal DNA degradation. Although commitment to cell death was induced by copper deficiency, the apoptotic process was partially inhibited as assessed from electrophoretic patterns of DNA degradation. Redifferentiation was closely linked to the apoptotic death program. Analysis of rescued detached cells in all three media (standard, Cu-, Fe-) indicated that the frequency of revertants was significantly higher among floating as opposed to adherent cell populations. Nevertheless, experimental copper depletion increased by 10(4) times the revertant frequency among adherent cells. We propose that redifferentiation of the variant hepatoma cells (and concomitant recovery of tumorigenicity) is determined by the gene expression pattern of programmed cell death.

Alterations of rat hepatoma cell genomes induced by copper deficiency.

Copper deficiency imposed on a variant rat hepatoma cell line inhibits cell growth and results in genesis of stable well-differentiated, tumorigenic revertants. The treatment caused a substantial increase in DNA content (up to 20%) of G1 and G2/M cells and inhibition of cell proliferation. This phenomenon was correlated with an enhancement of DNA replication. The excess DNA was unstable and rapidly lost with reinitiation of cell growth and mitosis. Minute and double-minute extrachromosomal material was detected by metaphase analysis, suggesting widespread DNA amplification in copper-deficient conditions. Although transitory, these genetic events were associated with genesis of drug-resistant cells and induction of tumorigenicity of the variant hepatoma cells. The data reveal a novel aspect of the consequences of trace element deficiency.

Extinction of peroxisomal functions in hepatoma cell-fibroblast hybrids.

Although peroxisomes are ubiquitous, differences in the number of organelles and in the expression of associated metabolic activities are observed, depending on the cell type. To investigate the control of peroxisomal activity in connection with cell differentiation, we constructed hybrids between two types of cells whose histogenetic origins dictate significant differences in peroxisomal activities: hepatoma cells and fibroblasts, with high and low expression, respectively, of peroxisomal functions. In these hybrids, extinction of the elevated activities that characterize liver cells is observed, in parallel with the well-documented extinction of differentiated functions. This suggests the existence in fibroblasts of a negative trans-acting regulation.

The inductive effect of a human DNA sequence (HALF1) on the differentiation of a variant rat hepatoma cell (C2) is restricted to episomal forms of the molecule.

HALF1, a 4.3 kb human DNA sequence, was originally identified as a double-stranded, closed-circular DNA molecule in revertants from a dedifferentiated rat hepatoma cell (C2) transfected with human liver DNA. Here we report its specific properties in inducing the transition to the hepatic phenotype. (i) In vitro recircularized HALF1 induces reversion after a minimum time lag of 7 days post-transfection. (ii) After induction, the presence of HALF1 is not required for maintaining the induced hepatic state. (iii) HALF1 is detected as a sequence integrated in high molecular mass DNA of human liver. (iv) HALF1 monomer or dimer plasmid constructs do not induce reversion when integrated into the genome of transfectants. (v) Short ubiquitous RNA transcripts (approximately 400 bases) are detected with specific HALF1 probes. These results indicate that the reversion process is linked to the presence of HALF1 extrachromosomal molecules.

Deleted chromosome 20 from a patient with Alagille syndrome isolated in a cell hybrid through leucine transport selection: study of three candidate genes.

Alagille syndrome (AGS) is a well-defined genetic entity assigned to the short arm of Chromosome (Chr) 20 by a series of observations of AGS patients associated with microdeletions in this region. By fusing lymphoblastoid cells of an AGS patient that exhibited a microdeletion in the short arm of Chr 20 encompassing bands p11.23 to p12.3 with rodent thermosensitive mutant cells (CHOtsH1-1) deficient in-leucyl-tRNA synthetase, we isolated a somatic cell hybrid segregating the deleted human Chr 20. This hybrid clone, designated NR2, was characterized by several methods, including PCR, with eight pairs of oligonucleotides mapped to Chr 20: D20S5, D20S41, D20S42, D20S56, D20S57, D20S58, adenosine deaminase (ADA), and Prion protein (PRIP); Restriction Fragment Length Polymorphism (RFLP) analyses with four genomic anonymous probes (D20S5, cD3H12, D20S17, D20S18); and fluorescent in situ hybridization (FISH) with total human DNA and D20Z1, a sequence specific to the human Chr 20 centromere, as probes. The NR2 hybrid allowed us to exclude three candidate genes for AGS: hepatic nuclear factor 3 beta (HNF3 beta), paired box 1 (PAX1), and cystatin C (CST3) as shown by their localization outside of the deletion. The NR2 hybrid is a powerful tool for the mapping of new probes of this region, as well as for obtaining new informative probes specific for the deletion by subtractive cloning of the region. Such markers will be useful for linkage analysis and screening of cDNA libraries.

Inductive effect of copper deficiency on the reversion of dedifferentiated rat hepatoma cells and on gene amplification.

Cells of a dedifferentiated rat hepatoma clone were submitted in vitro to copper deficiency. This treatment caused inhibition of cell growth. In addition, in treated cultures, the frequency of differentiated revertants selected in glucose-free medium was drastically increased when compared with the spontaneous frequency. The maximum effect was observed when cell proliferation spontaneously resumed after 20 days of copper deficiency. Furthermore, a copper depletion/replenishment protocol applied before the selection of revertants reduced the period of time of copper deficiency that was necessary to provoke the reversion process. It has been previously demonstrated that cell growth arrest and reinitiation may induce gene amplification events. Amplification of the dihydrofolate reductase gene as an indicator of such events was tested during the copper deficiency treatment. The frequency of cells resistant to increasing methotrexate concentrations due to gene amplification was enhanced by the treatment, just as was the frequency of differentiated revertants. These results suggest that in rat hepatoma cells the phenotypic transition to the stable differentiated state involves gene amplification and/or genome rearrangement.

Periodic fluctuations in proliferation of SV-40 transformed human skin fibroblast lines with prolonged lifespan.

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.

Analysis of the inductive effect of the genomic equivalent of HALF1 sequence in the reversion of rat dedifferentiated hepatoma cells.

HALF1 (human activator of liver function 1) is a closed DNA duplex implicated in reversion of rat dedifferentiated C2 hepatoma cells to a well-differentiated state. A copy of HALF1 is found in high-molecular-weight DNA in the human genome. The genomic equivalent of HALF1 and its flanking sequences [gH(5'-3') fragment] have previously been cloned and sequenced. To analyze the ability of the gH(5'-3') fragment to induce reversion process of C2 cells, two series of transfections were performed: (1) cotransfection of gH(5'-3') and plasmid pSVneo1 and (2) transfection of gH(5'-3') inserted into pSVneo1. The frequency of reversion was enhanced in transfected cells from the first experiment whereas no revertants were obtained from transfected cells in the second one. DNA analysis of the revertant clones revealed that reversion is associated with the transient presence of nonintegrated gH(5'-3') molecules. C2 cells were also transfected with gH(5'-3') cloned in pSV2dhfr. In the products of this transfection, the genesis of revertants correlated with amplification process of the gH(5'-3') sequence. We conclude that the presence of integrated copies of gH(5'-3'), even in high copy number, is not sufficient to induce the reversion process. We propose that extrachromosomal forms of gH(5'-3'), either given to cells or formed during amplification cycles, are involved in the reversion process of C2 cells.

Chromosomal localization and sequence analysis of a human episomal sequence with in vitro differentiating activity.

The genomic fragment carrying the human activator of liver function, previously described as an episome capable of inducing differentiation upon transfection into a dedifferentiated rat hepatoma cell line, was mapped on human chromosome 12q24.2-12q24.3. This chromosomal location was indistinguishable by in situ hybridization from that of the gene coding for the hepatic transcription factor HNF1. The sequence of the integrated form of the episome as well as its flanking sequences show that it is rich in retroposons. It contains a human ribosomal protein L21 processed pseudogene, one truncated L1Hs sequence, and 10 Alu repeats, which belong to different subfamilies.

The human episome HALF1: structure of its genomic counterpart.

A human episomal sequence (HALF1) has been identified by its ability to restore expression of hepatic functions when used to transfect a rat dedifferentiated cell line. The genomic equivalent of this human episome (gHALF1) and its flanking sequences were analyzed. HALF1 itself does not present the characteristics of a transposable element but half of its sequence corresponds to retroposons, including Alu and L1 repeats and a processed pseudogene, known to transpose via RNA intermediates. The structural characteristics of these different kinds of retroposons and their origin and evolution were analyzed.

Resistance to erucic acid as a selectable marker for peroxisomal activity: isolation of revertants of an infantile Refsum disease cell line.

A system based on the ability of cells to oxidize very long-chain fatty acids (VLCFA) was developed to select in vitro normal human fibroblasts from fibroblasts of patients suffering from peroxisomal disorders with multienzymatic deficiencies: Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum disease (IRD). Cells treated with various concentrations of erucic acid (C22:1 n-9) revealed an enhanced toxicity of this fatty acid for the fibroblasts of patients compared with normal cells. This differential toxicity is correlated with variable accumulations of C22:1 n-9 and the absence of beta-oxidation products in the mutants. Revertants from clonal IRD cell lines were isolated in the selective medium at frequencies ranging from 3 x 10(-7) to 4 x 10(-6) depending on the line. After six weeks of growth in the absence of selective pressure, the variants exhibited a resistance level to C22:1 n-9 identical to that of normal cells. Furthermore, beta-oxidation of VLCFA is re-established in these selected cells as well as dihydroxyacetone phosphate acyltransferase activity. Immunoblot experiments also demonstrated a restored pattern of acyl-CoA oxidase molecular forms. Last, immunofluorescence studies revealed the presence of cytoplasmic structures that were absent in the original IRD cells. Thus, both the deficiencies in metabolic pathways and paucity of the organelle are at least partially corrected in the selected clones.

Reversion and cell hybridization of a dedifferentiated rat hepatoma cell: dissociation of mechanisms establishing the hepatic traits.

The expression of a mouse serum albumin (MSA) gene introduced into differentiated and dedifferentiated variant cells of a rat hepatoma line depends on the overall differentiated state of the recipient cell. Thus, the transcription rate of this mouse gene is very high in differentiated cells compared to that in the variant. Furthermore, the activation of its expression is observed during the course of reversion of the variant cell. In well-differentiated hybrids between the variant and cells of the original line, the expression of the MSA gene depends on the phenotype of the transfected parental cell: transcription of this foreign gene is detected only in hybrids in which it was initially integrated into the genome of the differentiated parent. Gene dosage effects are not involved as the copy number of the MSA gene is identical in both types of transfected cells. These results demonstrate that the establishment of the liver phenotype in differentiated hybrids and revertants is due to different mechanisms.

Update on genetic and molecular investigations of diseases with general impairment of peroxisomal functions.

A group of genetically determined peroxisomal diseases is characterized by both multiple enzymatic deficiencies and abnormal structural features of the organelle. The primary cause of the phenotypes is likely to involve peroxisome assembly impairment. Complementation analyses performed on fibroblasts of patients revealed the existence of at least eight groups that do not reflect the clinical classifications. Recently, the use of experimental models led to the identification of a gene encoding for a peroxisomal membrane protein (PAF-1) in which a mutation was associated with the altered phenotype in a complementation group of the Zellweger syndrome (paradigm of these diseases). Also revealed in Zellweger probands are mutations of a gene encoding another peroxisomal protein (PMP70).