Effect of immobilized laminin on karyotypic variability in two karyotypically different variants of the indian muntjac skin fibroblast cell line

The numerical and structural karyotypic variability has been investigated in MTs of the markerless cell line of Indian muntjac skin fibroblasts, as well as in its karyotypic variant MT^D cultivated on a laminin 2/4 coated surface. In the MT cell line preincubated in serum-free medium for 2.5 and 1.0 h, then cultivated on a laminin-coated surface in serum-containing medium for one, two, and three days, the character of cell distribution for the chromosome number has changed. These changes involve a significant decrease in the frequency of cells with modal numbers of chromosomes and an increase in frequency of cells with lower chromosome numbers. Some new additional structural variants of the karyotype (SVK) appeared. The observed alterations seem to be due to disturbances of the chromosome segregation and the establishment of a new advantageous balanced karyotypic structure. In the karyotypic variant MT^D differing from MT by an increased number of dicentrics (telomeric associations) cultivated under the same conditions, the character of cell distribution for the chromosome number did not change. In the MT cell line, the frequency of chromosomal aberrations did not change relative to control variants. In the karyotypic variant MT^D under the same conditions, the frequency of chromosomal aberrations significantly increased after three days mainly due to the formation of dicentrics. These results confirm the conclusion that, like aneuploidy, the formation of dicentrics in markerless cell lines appears to be the way in which the cell population adapts to unfavorable environmental factors. Possible reasons for differences in the character of the numerical and structural karyotypic variability between the MT cell line and its karyotypic variant MT^D are discussed.     

The effect of immobilized fibronectin on karyotypic variability in the skin fibroblast cell subline of indian muntjac

The numerical and structural karyotypic variability has been investigated in the Indian muntjac skin fibroblast cell subline MT on cultivating cells on the fibronectin-coated surface. In cell subline MT, cultivated on the fibronectin-coated surface for 1 and 2 days, the character of cell distribution for the chromosome number did not change. In 3, 4 and 8 days, the character of cell distribution for the chromosome number changed. These changes involve a significant decrease in frequency of cells with modal numbers of chromosomes, and an increase in frequency of cells with lower chromosome numbers. Many new additional structural variants of the karyotype (SVK) appear. The observed alterations seem to be due to both disturbances of mitotic apparatus and selection of SVK, which are more advantageous to changed culture conditions of the cell population. Detachment of cells from the fibronectin-coated surface, followed by a 1 day cultivation on a hydrophilic surface, commonly used for routine cell cultivation, does not restore the control cell distribution for the chromosome number, but cultivation in these conditions for 5 days restore control distribution. The frequency of chromosomal aberrations on cultivation on the fibronectin-coated surface for 3 and 4 days significantly increases, mainly at the expence of dicentrics (telomeric association). On prolongating the time of cultivation up to 8 days on the fibronectin-coated surface the frequency of chromosomal aberrations approaches the control value. Structural instability of chromosomes at cultivation on the fibronectin-coated surface demonstrates nonspecific reaction of "markerless" cell lines to unfavourable factors of the environment. We discuss possible reasons of differences in the character of karyotypic variability in cell lines of the Indian muntjac skin fibroblasts on cultivating on laminin and fibronectin.     

The influence of a substrate including extracellular matrix proteins on karyotypic variability in two cell lines of Indian muntjac skin fibroblasts

The influence of cell-culture conditions on numerical and structural karyotypic variability has been investigated in two Indian muntjac skin fibroblast “markerless” cell lines, M and MT. The cells were cultivated on a substrate consisting of extracellular matrix proteins (ECMs) synthesized by human mesenchymal stem cells (SC5-MSC). The character of cell distribution for the chromosome number of the cell line M changed after cultivation for 1 and 4 days as compared to control cells, which were cultured on a hydrophilic surface without ECM coating. These changes involve a significant decrease in frequency of cells with the modal numbers of chromosomes and an increase in frequency of cells with lower chromosome numbers. Many new types of additional structural variants of the karyotype (SVK) appear. The MT cell line, differing from M line in the number of homologous chromosomes, exhibited a character of cell distribution similar to that of the M line for the chromosome number for only 1 day after cultivation on the ECM substrate, but not after 4 days under the same culture conditions, when no difference from the control cells was observed. Further cultivation of MT cells for 8 days did not change the character of cell distribution for the chromosome number relative to the control variant. The observed alterations seem to be due to disturbances in the correct chromosome-segregation process, which were caused by an abrupt shift in the cell-culture conditions. Analysis of the structural karyotypic variability revealed a significant increase in frequency of chromosomal aberrations in the M cell line for 1 and 4 days in culture on the ECM substrate as compared to the control cells. The frequency of dicentric chromosomes (telomeric associations) was increased and constituted more than 50% of all chromosome aberrations. No increase in frequency of chromosome aberrations was observed for MT cells cultured under the same conditions. It can be suggested that the differing by the karyotypic structure, but the genetically identical cell lines have different response to the substrate. In contrast to the M line, in the MT line, a fast normalization of numerical karyotypic characteristics and no enhancement of structural karyotypic variability takes place. This provides a possibility to cultivate an MT cell on the given protein substrate while maintaining a balanced karyotypic structure characteristic of MT cell line.     

The influence of immobilized fibronectin on karyotypic variability of two rat kangaroo kidney cell lines

The numerical and structural karyotypic variability has been investigated in "markerless" Rat kangaroo kidney cell lines NBL-3-17 and NBL-3-11 when cultivating on a fibronectin-coated surface. In cell line NBL-3-17, cultivated on the fibronectin-coated surface for 1, 2, 4 and 8 days, the character of cell distribution for the chromosome number has changed. These changes involve a significant decrease in frequency of cells with modal number of chromosomes, and an increase in frequency of cells with lower chromosomal number. Many new additional structural variants of the karyotype (SVK) appear. The observed alterations seem to be due preference adhesion of cells with lower chromosome number, disturbances of mitotic apparatus and selection of SVK, which are more adopted to changes in culture conditions. Detachment of cells from the fibronectin-coated surface, followed by 5 days cultivation on a hydrophilic surface restored control distribution. In cell line NBL-3-11, cultivated on the fibronectin-coated surface for 1, 2, 4 and 8 days, the character of numerical karyotypic variability did not change compared to control variants. In cell line NBL-3-17 the frequency of chromosomal aberrations under cultivation on the fibronectin-coated surface for 1, 2, 4 and 8 days did not change relative to control variants. In cell line NBL-3-11 the frequency of chromosomal aberrations under the same conditions significantly increases, mainly at the expence of chromosomal, chromatid breaks and dicentrics (telomeric association) relative to control variants. We discuss possible reasons of differences in the character of numerical and structural karyotypic variability between cell lines NBL-3-17 (hypotriploid) and NBL-3-11 (hypodiploid) under cultivation on fibronectin. The reasons of the observed interline karyotypic differences possibly consist in peculiarity of karyotypic structure of cell line NBL-3-11 and in the change of gene expression, namely in a dose of certain functioning genes in the hypotryploid cell line NBL-3-17.     

Karyotypic variability in rat kangaroo cell lines cultivated on fibronectin-coated surface

Numerical and structural karyotypic variability was investigated in the NBL-3-17 and NBL-3-11 “markerless” rat kangaroo kidney cell lines cultivated on a fibronectin-coated surface. For the NBL-3-17 cell line grown on a fibronectin-coated surface for periods of 1, 2, 4 and 8 days, the chromosome number distribution changed. These changes involved a significant decrease in the frequency of cells with the modal chromosome number and an increase in the frequency of cells with a lower chromosome number. Many new additional structural variants of the karyotype (SVK) appeared. The observed alterations seem to be due to the predominant adhesion of cells with a lower chromosome number, disturbances of the mitotic apparatus and selection for SVK adapted to the changes in culture conditions. Detachment of cells from the fibronectin-coated surface followed by 5 days cultivation on a hydrophilic surface restored the control cell distribution. For the NBL-3-11 cell line cultured on the fibronectin-coated surface for 1, 2, 4 and 8 days, the numerical karyotypic variability did not change compared to control variants. For the NBL-3-17 cell line grown on a fibronectin-coated surface for 1, 2, 4 and 8 days, the frequency of chromosomal aberrations also did not change relatively to the control. In the NBL-3-11 cell line, the frequency of chromosomal aberrations under the same conditions significantly increased, mainly due to chromosome and chromatid breaks and dicentrics (telomeric associations). The differences in the numerical and structural karyotypic variability between NBL-3-17 (hypotriploid) and NBL-3-11 (hypodiploid) cell lines cultivated on fibronectin are discussed. It is assumed that the observed differences in the karyotypic variability between these cell lines were determined by the specific karyotypic structure of the NBL-3-11 cell line and the altered gene expression of the NBL-3-17 hypotriploid cell line caused by increased doses of certain functioning genes.     

Effect of laminin on structural karyotype variability of kangaroo rat kidney cell lines

The structural karyotypic variability has been investigated in the "markerless" epithelial-like Rat kangaroo kidney cell lines NBL-3-17 and NBL-3-11 on cultivation on a laminin-2/4 coated surface. In cell line NBL-3-17, cultivated on the laminin-coated surface for 2, 4 and 12 days, and in cell line NBL-3-11, cultivated on the laminin-coated surface for 2 and 4 days, there is a significant increase in the frequency of chromosomal aberrations, both chromosomal breaks and dicentrics (telomeric associations). Different sensitivity of individual chromosomes to inducing chromosomal breaks was observed in addition to a preferential involvement of some chromosomes in dicentric formation. Structural instability of chromosomes at cultivation on laminin demonstrates nonspecific reaction of the "markerless" cell lines to unfavourable factors of the environment. We discuss possible reasons of differences in the character of karyotypic variability between a cell line of the Indian muntjac skin fibroblasts and epithelial-like Rat kangaroo kidney cell lines cultivated on laminin.     

Effect of laminin on numerical karyotype variability of kangaroo rat kidney cell lines

The numerical karyotypic variability has been investigated in "markerless" epithelial-like Rat kangaroo kidney cell lines NBL-3-11 and NBL-3-17 on cultivation on a laminin-2/4 coated surface. In cell line NBL-3-17, cultivated on the laminin-coated surface for 2, 4 and 12 days, the character of numerical karyotypic variability has changed. In 2 days the general character of cell distribution for the chromosome number did not change, but the frequency of cells with modal number of chromosomes decreases significantly, while that of cells with lower chromosome number show a tendency to increase. At a prolongation of cultivation time to 4 and 12 days, the numerical karyotypic heterogeneity in cell population increases due to a significant change in the general character of cell distribution for the chromosome number, which is caused by a significant decrease in the frequency of cells with the modal number of chromosomes, and by an increase in the frequency of cells with lower chromosome number. The analysis of distribution of individual chromosomes showed that the number of types of additional structural variants of the karyotype (SVK) increases significantly on cultivation on laminin for 2-12 days. In cell line NBL-3-11, cultivated on the laminin-coated surface for 2 and 4 days, the character of numerical karyotypic variability did not change compared to control variants. Possible reasons of the observed changes of numerical karyotypic variability in cell line NBL-3-17 is discussed. The reason of differences in the character of numerical karyotypic variability between cell lines NBL-3-11 and NBL-3-17 possibly consists in the change of gene expression, namely in a dose of certain functioning genes. The polymerase chain reaction with arbitrary primers revealed no differences between DNA patterns of cell lines NBL-3-17 and NBL-3-11. This can reflect a similarity in the primary DNA structure of both cell lines. Hence, these lines differ only in the number of homologous chromosomes (hypotriploid and hypodiploid).     

Effect of <Emphasis Type="Italic">Mycoplasma salivarium</Emphasis> with and without L-arginine on karyotypic variability in cell line of Indian muntjac skin fibroblasts at long-term cultivation

The effect of Mycoplasma salivarium on the numerical and structural karyotypic variability was studied in the markerless cell line of Indian muntjac skin fibroblasts (line M) during long-term cultivation with and without L-arginine. The cultivation of mycoplasma-contaminated cells for 15 and 30 days did not change the character of cell distribution for the number of chromosomes. In contaminated cells cultivated for 60 and 75 days, the character of cell distribution for the chromosome number changed. These changes involve bimodal distribution for the chromosome number due to a significant decrease in frequency of the cells with the modal number of chromosomes with the main structural variant of karyotype (SVK) 2 + 2 + 1 + 1 + 1 and an increase in the frequency of cells with the submodal number of chromosomes with a main SVK of 2 + 2 + 1 + 1. Furthermore, a significant increase in the frequency of cells with lower numbers of chromosomes was observed after 60 days compared to that after 75 days of cultivation. After the cultivation of the contaminated and control cells in the medium with an elevated concentration of L-arginine for 60 days, the numerical parameters were unchanged relative to the control. The cultivation of contaminated cells for 60 days followed by the addition of L-arginine for 15 days restored the numerical parameters to the control level. In the contaminated cells, the frequency of chromosomal aberrations for 30, 60, and 75 days increased significantly compared to the control variant. After 30 days of cultivation, a small but statistically significant increase took place due to a uniform slight increase in the frequency of chromosomal aberrations of all types. After 60 and 75 days, a greater increase occurred due to a statistically significant increase in the frequency of chromosomal and chromatid breaks. Moreover, after 60 days, the level of dicentrics (telomeric associations) mainly produced by chromosomes 1 and 2 increased significantly. The role of dicentrics as for a means of adaptation for markerless cell lines to the condition of cultivation and the role of L-arginine in the restoration of the normal karyotypic structure of the line M cell population at mycoplasmal contamination are discussed.     

The influence of Mycoplasma salivarium in the absence and presence of L-arginine on karyotypic variability in cell line of the Indian muntjak skin fibroblasts under long-term cultivation

The influence of Mycoplasma salivarium on the numerical and structural karyotypic variability has been investigated in the "markerless" cell line of the Indian muntjak skin fibroblasts (line M) during long-term cultivation in the absence and presence of L-arginine. Cultivation of the mycoplasmal contaminated cells for 15 and 30 days did not change the character of cell distribution for the chromosome number. In the contaminated cells cultivated for 60 and 75 days, the character of cell distribution for the chromosome number was changed. These changes involved bimodal distribution for the chromosome number due to a significant decrease in the frequency of the cells with the modal number of chromosomes with main structural variant of karyotype (SVK)--2 + 2 + 1 + 1 + 1 and an increase in the frequency of cells with submodal number of chromosomes with main SVK--2 + 2 + 1 + 1. Besides, a significant increase in the frequency of the cells with lower chromosome number was observed in 60 days compared to that in 75 days of cultivation. Cultivation of the contaminated and control cells in the medium with increased concentration of L-arginine during 60 days did not change the numerical parameters relative to the control. Cultivation of the contaminated cells for 60 days followed by addition of L-arginine for 15 days restored the numerical parameters the numerical parameters to the control level. In the contaminated cells the frequency of chromosomal aberrations significantly increased for 30, 60 and 75 days cultivation relative to the control variant. In 30 days, the small but significant increase took place due to increase in the frequency of chromosomal aberrations of all the types. In 60 and 75 days, a greater increase took place due to a significant increase in the frequency of chromosomal and chromatid breaks. Moreover, in 60 days, the level of dicentrics (telomeric associations) mainly produced by chromosomes 1 and 2 increased significantly. The role of dicentrics as one of the ways for adaptation of the "markerless" cell lines to condition of cultivation and the role of L-arginine in the restoration of normal karyotypic structure of cell population of line M under mycoplasmal contamination are discussed.     

Effect of laminin on karyotypic variability in cell line of Indian muntjac skin fibroblasts

The numerical and structural karyotypic variability has been investigated in the Indian muntjac skin fibroblasts cell line M and karyotypic variant of this line M' on cultivation on a laminin 2/4 coated surface. In cell line M, cultivated on the laminin-coated surface for 4 and 14 days, and in karyotypic variant M', cultivated for 2, 4 and 14 days, the character of cell distribution for the chromosome number has changed. These changes involve a significant decrease in frequency of cells with model numbers of chromosomes, and an increase in frequency of cells with lower chromosome numbers. As a result, new modal chromosome numbers form. The frequency of cells with 4 chromosomes increases significantly; as a rule, such cells are absent in the control cell variants. Many new additional structural variants of the karyotype (SVK) appear. Detachment of cells M' from the laminin-coated surface followed by a 2 day cultivation on a hydrophilic surface, commonly used for routine cell cultivation, does not restore the control cell distribution for chromosomal number. The frequency of chromosomal aberrations on cultivation of the laminin-coated surface does not change relatively to controls. The observed alterations seem to be due to both disturbances of mitotic apparatus and selection of SVK, which are more advantageous to changed culture conditions of the cell population.     

Effect of mycoplasma contamination of a human cervical carcinoma cell line M HeLa clone 11 on karyotypic variability

The karyotypic variability has been investigated for an immortalized human epithelioid cervix carcinoma cell line M HeLa clone 11, cultivated for 15-60 days after contamination with Acholeplasma laidlawii A, strain PG-8, and for 30 days after contamination with Mycoplasma arginini R-16. The character of cell distribution for chromosome number changes in contaminated cells significantly, as compared to the control. So, the frequency of cells with the modal number of chromosomes being equal to 50 decreases significantly, and the range of variability in the number of chromosomes increases. With the prolongation of the term of cultivation in control variants up to 60 days the character of cell distribution for chromosomal number changes, as compared to the preceding terms (15 and 30 days), which is expressed in the extended range of variability in the chromosomal number at the expense of decreased frequency of cells with submodal number of chromosomes equal to 49. But the degree of these changes is significantly smaller than in contaminated variants. The frequency of polyploid cells did not differ in all investigated variants. The number of chromosomal aberrations in cultures contaminated with A. laidlawii (for 15-60 days) and M. arginini (for 30 days) does not differ from that in the corresponding controls. The absence of dicentrics (telomeric association) at a long-term contamination of the human epithelioid cervix carcinoma cell line M HeLa clone 11 having marker chromosomes in karyotype and a comparison of these results with the earlier obtained data on other "marker" and "markerless" cell lines seems to confirm the point of view that dicentrics appear a characteristic feature of karyotypic variability of "markerless" cell lines, mainly with a long-term contamination in different conditions.     

Characteristics of quantitative variability of karyotype in cell line of fibroblasts from indian muntjac

The numerical regularities of karyotypic variability in cell line of the Indian muntjac skin fibroblasts (Muntiacus muntjak) has been studied. It was found that the karyotypic structure of cell population is mainly determined by some number of specific variant deviations from the main structural variant of karyotype (MSVK) to be depended on internal connections between chromosomes. Specific regulations determining the karyotypic structure of cell population are: 1) nonrandom character of cell distribution according to the number of chromosomal deviations from MSVK; 2) specific character of deviations in each chromosome from MSVK; 3) presence of significant connections between individual chromosomes by simultaneous mainly single directed numeral deviations. Results presented in this investigation were thought owner by analysing the number of individual chromosomes. These results extend considerably the known ideas of regulations of karyotypic variability in cell populations in vitro.     

The effect of mycoplasmal contamination of human embryonic lung cell line MRC-5 on the karyotypic variability

Karyotypic variability has been investigated for nonimmortalized human embryonic lung cell line MRC-5, cultivated with Acholeplasma laidlawii strain PG-8 for 15-45 days. The character of cell distribution for chromosome number did not change during this time. In all investigated variants the number of polyploid cells increased considerably with the lengthening of the term after decryoconservation. The number of chromosomal aberrations in 15-45 days contaminated cells increased significantly as compared to the control at the expense of dicentrics (telomeric associations). The number of dicentrics had a tendency to increase with the lengthening of the term of contamination. Thus, in 45 days the number of dicentrics increased twice as much as that in 15 days. The increase of polyploids may be due presumably to the specific character of karyotypic variability in nonimmortalized cell lines with the long-term cultivation. Our present and previous results made it possible to suppose that the formation of dicentrics (telomeric associations) in nonimmortalized "markerless" cell line, following the long-term mycoplasmal contamination, may prove additionally the role played by dicentrics in cell adaptation to in vitro conditions whatever the degree of transformation may be--nonimmortalized line or immortalized nontumorogenic or high tumorogenic lines.     

Karyological characteristics of cell sublines of the kidney of the kangaroo rat and of skin fibroblasts of the Indian muntjac

A study was made of the karyotypic structure of sublines derived from the kangaroo rat's kidney (NBL-3) and skin fibroblasts of the Indian muntjac, available in the cell culture bank of the Institute of Cytology Acad. Sci. USSR. A comparative karyologic analysis was made of subline NBL-3 both contaminated with mycoplasma (NBLK) and decontaminated with antibiotics (NBLD). Authentic differences in cell distribution according to chromosome number in NBLK and NBLD variants were shown. Modal numbers of chromosomes are 11 and 17, respectively. The modal number for the Indian muntjac cell subline (MT) is 9. 60-80% of the cells had an identical karyotype (the main structural variant of the karyotype is MSVK). Using the G-banding technique, all the MSVK variants were shown to display constant karyotypes. In NBLK there are 5 pairs of homologous chromosomes and one metacentric. In NBLD, the number of homologous chromosomes increased in all the groups (hypotriploid karyotype). In subline MT 3 homologous chromosomes were found in groups I and IV, 2 in group III in addition to one X-chromosome. A comparison with the Indian muntjac karyotype showed the absence of marker chromosomes in MT. The analysis of additional SVK shows that the deviations from MSVK are caused mostly by changes in the number of homologous chromosomes within the groups. A study of the frequency of deviations in chromosome numbers observed in the groups from MSVK showed that different chromosomes were involved in karyotypic changes in the same way in the "low-chromosome" variants of NBLK and MT, and in different ways in NBLD. A comparison of the "premycoplasmic" variants of line NBL-3 with NBLK shows no differences in the parameters studied.(ABSTRACT TRUNCATED AT 250 WORDS)     

Karyotypic variability of human multiple myeloma cell lines

Cytogenetic analysis of human multiple myeloma (MM) cell lines L363, Karpas 707, RPMI 8226, and U-266 was carried out. During long-term existence in vitro, the number of chromosomes in the cell lines was shown to be preserved at the near-diploid level (L363, Karpas 707, U-266) or to increase up to the hypotriploid level (RPMI 8226). There were complexly rearranged karyotypes with abnormalities of chromosomes of all pairs in all cell lines; however, no identical chromosomal translocations have been revealed. Loci of chromosomes involved in structural rearrangements in these cell lines often coincided with sites of DNA copy number imbalances characteristic for MM in vivo. Distinct types of the karyotypic structure of cell populations differing in the combination of cells with the main and additional structural variants of karyotype and of cells with nonclonal chromosome rearrangements were found in MM cell lines. In general, the karyotypic variability of the MM cell lines corresponds to the dynamics of karyotype of myeloma cells in vivo and, hence, has a tumor-specific character.     

Effect of cultivation conditions on the karyotype structure of a cell subline of the kangaroo rat kidney

Variations in cultivation conditions were found to exert influence on the distribution of cells for chromosome number by changing the modal class. The change of the HMEM medium for the EMEM medium during 2-6 passages results in the appearance of a new modal class with 16 chromosomes. The change in the chromosome number is preferably due to the loss of one X chromosome within the main structural variant of the karyotype (MSVK). On the other hand, the change of the HMEM medium for the F12 medium during 4-6 passages does not affect the cell distribution for the chromosome number. A comparative analysis of the total frequency of the MSVK cells and that of MSVK cells of the modal class showed that the karyotypic changes took place in all the variants, both in the modal class and beyond it due to other additive SVK. An exception is the variant NBLD (change of HMEM for the F12 during 6 passages). In this case chromosome changes occur mostly in the modal class, primarily due to the redistribution of chromosomes in groups. In all the variants there is an insignificant frequency of chromosomes, morphologically different from the MSVK. This confirms the findings according to which chromosomal changeability in the NBLD may be associated mostly with the change in the number of homologous chromosomes rather than with chromosomal aberrations. The frequency of chromosomal aberrations is the same in all the variants examined. The dependence of karyotypic characteristics on culture media mentioned above indicate that care should be taken in choice of culture conditions for permanent cell lines.     

The effect of culturing conditions on the karyotypic structure of two cell sublines of Indian muntjak skin fibroblasts

The "therapeutic" doses of antibiotics, routinely applied to prevent microbial contamination in cultured cells, decrease the frequency of modal class cells and increase that of cells of other classes in sublines of Indian muntjak skin fibroblasts. In MT-subline, with 9 chromosomes in the modal class, the loss of cells with some large chromosomes occurred almost frequently. In terms of the formula of the karyotype main structural variant, this change is described as (-1-0-1-1). In M-subline, with 7 chromosomes in the modal class, the similar result is mainly achieved due to a decrease in the cell number with Y1-chromosome to be described as (0-0-0-0-1). The study of frequency of deviation from the chromosome number in the MSVK has shown that in the MT-subline, rather than in the M-subline, different chromosomes are incidentally involved in the karyotypic rearrangement. In both the sublines antibiotics induced chromosomal aberrations, primarily increasing the number of dicentrics. Preferential involvement of some chromosomes in the dicentric formation was observed. Cytogenetical parameters are more affected by antibiotics in the MT-subline. The data obtained indicate that even low concentrations of antibiotics may induce karyotypic changes in cells cultures.     

Epidermal growth factor receptor expression related to differentiation capacity in normal and transformed keratinocytes

Epidermal growth factor (EGF) and Ca2+ have been indicated to play a major role in skin development. We have used normal keratinocytes, SV40-transformed keratinocytes (SVK14) and various squamous carcinoma cell (SCC) lines as in vitro model system to study the effect of the extracellular Ca2+ concentration of EGF-receptor expression in relation to the capability of cells to differentiate. The cell lines used exhibit a decreasing capacity to differentiate in the order of keratinocytes approximately SVK14 greater than SCC-12F2 greater than SCC-15 greater than SCC-12B2 greater than SCC-4, as judged from Ca2+-ionophore-induced cornified envelope formation. Under normal Ca2+ conditions, all cell lines (except for SCC-15) exhibited two classes of EGF-binding sites. The number of low-affinity binding sites increased considerably as cells were less able to differentiate, while the apparent dissociation constant (kd) was similar in all cell lines. In contrast, the properties of high-affinity EGF binding varied in the various cell lines without a clear relationship to the degree of differentiation capacity. Lowering the extracellular Ca2+ concentration to 0.06 mM resulted in a decrease of Ca2+ ionophore-induced cornified envelope formation, demonstrating the decreased ability to differentiate under these conditions. The decreased ability to differentiate was accompanied by a marked increase in the number of EGF-binding sites, but without a change of the kd. Furthermore, no high-affinity EGF-binding sites were detectable under these conditions. Finally, addition of Ca2+ to low Ca2+-cultured cells caused a rapid decrease of EGF binding in all cell lines, most prominently in normal keratinocytes and SCC-12F2 cells. The data presented demonstrate: The combination of normal keratinocytes, SVK14 and the various SCC lines provides an attractive model system to study differentiation in vitro; EGF-receptor expression is related to the state of differentiation, both phenomena being sensitive to the external Ca2+ concentration; and EGF-receptor expression is related to the capability of cells to differentiate.     

Characteristics of quantitative karyotypic variability in cell line of kidney from rat kangaroo (Potorous tridactylis)

The goal of this work was to investigate the numeral karyotypic variability in different sublines (MT, M2). These sublines are formed spontaneously from the main cell line (M) and have modal number of chromosomes 9 and 10, MSVK (main structural variant karyotype)--3 + 3 + 1 + 2 and 3 + 4 + 2 + 1. There are general regulations which were originally got for the line M. In particular: 1) nonrandom character of cell distribution according to the number of chromosomal deviations from MSVK; 2) specific character of deviations of each chromosome from MSVK; 3) presence of significant connections between separate chromosomes by simultaneous, mainly single directed numeral deviations. These three lines (M, MT, M2) were compared and some differences were found: 1) different frequencies of deviations from MSVK; 2) the same chromosomes have tendency to different numeral deviation; 3) the specificity of some significant connections between separate chromosomes by simultaneous numeral deviations. These results lead us to a conclusion that the balance of numerical karyotypic structure in cell populations depends on the regulations connected with the character of deviations according to the number of chromosomes from MSVK which has the largest selected advantage. Each line has its own specific limits of karyotypic variability.     

Karyotypic stability of Serum-Free Mouse Embryo (SFME) cells

Mouse embryo cultures derived in serum-containing medium undergo growth crisis or senescence after fewer than 20 population doublings, followed by the emergence of genetically altered, polyploid immortalized cells capable of growing indefinitely. Serum-free mouse embryo (SFME) cells, derived in medium in which serum is replaced with growth factors and other supplements, do not exhibit growth crisis or gross chromosomal aberrations when cultured for well over 100 population doublings and display other unique properties. We examined culture conditions and physiological factors affecting karyotypic stability in long term cultures of SFME cells derived from several mouse strains. Cloning SFME cells consistently isolated colonies with altered karyotype, even when the clones were derived from parent cultures with no karyotypic alterations. After 140–200 population doublingsin vitro, the percentage of SFME cells showing hyperdiploidy or structural chromosomal abnormalities increased, although the modal chromosome number remained diploid. SFME cells transformed with molecularly cloned oncogenes did not show alterations in karyotype beyond that expected from the clonal origins of these cells, indicating that malignant transformation of SFME cells does not result in general karyotypic instability.