x chromosome inactivation and sv40 transformation of mammalian cells

Somatic Cell Genetics, Vol. 5, No. 6, 1979, pp. 945-955

X Chromosome Inactivation and SV40 Transformation of Mammalian Cells

Wendy H. Raskind and Stanley M. Gartler

Departments of Genetics and Medicine, University of Washington, Seattle, Washington 98195

Received 16 July 1979

Abstract--Five embryonic mouse cultures and one human fibroblast culture were transformed with SV40. The cultures were studied cytologically to see i f the normal pattern o f sex chromosome replication was maintained in SV40 transformed cells. Characteristic late replication patterns were observed for both the X and Y chromosomes, and there was no evidence for loss o f the inactive X chromosome, even in cells with 4 or more X chromosomes. The human line was heterozygous at two X-linked loci and a clonal analysis showed that the expression o f X-linked genes was not affected by S V40 transformation.

INTRODUCTION

One of the important questions about X chromosome inactivation (XCI) that remains to be clarified fully is that of permanence. The early observa- tions on variegation and clonal expression in heterozygotes gave strong support to the concept that once inactivation had occurred, the pattern of XCI became a fixed part of the cells' somatic heredity (1-3). More recent work indicates that under certain conditions reactivation of X-linked genes may occur in somatic cells (4, 5). In mice heterozygous for Cattanachs' translocation and associated markers, Cattanach has reported apparent progressive reactivation of the autosomal loci on the translocation chromosome in some cells of aging mice (4). In human-mouse somatic cell hybrids Kahan and DeMars (5) have shown that the hypoxanthine guanine phospho- ribosyltransferase (HGPRT) gene may reactivate at low frequencies. In marsupials where XCI is nonrandom, the paternal glucose-6-phosphate dehydrogenase (G6PD) gene may be reactivated when skin biopsies are grown in cell culture (6).

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0098-0366/79/1100-0945503.00/0 �9 1979 Plenum Publishing Corporation

946 Raskind and Gartler

Simian virus (SV40) treatment of mammalian cells is widely used as a means of achieving cell transformation. SV40 transformation affects cell morphology, growth, chromosome structure, segregation and replication, culture longevity, and gene expression (7-10). It has also been reported that SV40-transformed cells may exhibit preferential loss of X chromosomes (11, 12). In the course of studies on gene dosage and mutation induction' (13), we transformed a number of cell strains with SV40. Sex chromosome replication and X-linked gene expression were examined in these cultures and the results of these studies are the basis of this report.

MATERIALS AND METHODS

Cell Culture and Cell Lines. Cultures were begun from minced whole embryos of 15-16 days' gestation. At the first or second transfer from each explant, flasks were allocated for spontaneous and viral transformation. For viral transformation, SV40 virus (500-1000 PFU/cell) was added to a culture for 18-24 h. Culture procedures routine in this laboratory were used for all subsequent work (13-15). Cells were assayed for the SV40 TAg by a sandwich technique. Cells growing on coverslips were fixed with ice cold methanol, air dried, and incubated at room temperature with hamster anti-T antiserum (Flow lot TB 40051). The coverslips were then washed and incubated for 2 h in the dark with FITC-tagged rabbit anti-hamster antiserum and scored for the presence of nuclear fluorescence. Immunofluorescent staining of the SV40-transformed cultures, both mouse and human, was positive, whereas the spontaneously transformed cultures were T-antigen negative.

Cytology. Chromosome preparations, karyotyping, and autoradiography were carried out as previously reported (13).

Flow Microfluorimetry. Logarithmically growing cultures were har- vested with minimal exposure to trypsin. After centrifugation at 200g for 10 min at 5~ the cells were resuspended in 1 ml of cold saline, fixed with the addition of 3 ml cold 95% EtOH, and refrigerated until ready for assay (0-7 days). Prior to analysis of DNA content on the Flow Microfluorimeter (16), cells were centrifuged and stained in 1 ml mithramycin (Phizer, 0.1 mg/ml + 60 mM MgC12 in saline) for 2 h at room temperature.

Isozyme Studies. PGK (phosphoglycerate kinase) and G6PD electrophoretic analyses of clones of an SV40-transformed human cell line were carried out as previously reported (14).

RESULTS

Transformation of Primary Mouse Cells by SV40 Virus. Fifteen cultures, each begun from an individual embryo from a total of 3 litters, were

X Chromosome Inactivation and SV40 Transformation 947

explanted and grown for 6 days. Each culture was then divided in half, one part to be SV40 transformed. Following SV40 infection, the cells were not observed to undergo a growth crisis as occurs in mouse fibroblast cultures, but continued to require frequent passage to maintain a subconfluent density. By 3~, weeks 12 of the SV40-infected cultures exhibited a changed morpholo- g y - t h e cells being larger than their precursor fibroblasts. The remaining 3 cultures sloughed cells and never regained a healthy growth pattern. It is possible that these cultures suffered abortive transformation (17, 18). At various intervals, aliquots from the SV40-infected and -noninfected paired cultures were tested for ability to grow at low density. At 21/2-4 weeks the SV40-infected cultures exhibited this property with varying success. Cloning efficiency improved with time of culture, increasing in one line from approximately 0.7% at 4 weeks, 4% at 5 weeks, 8% at 2 months, to about 31% after 18 months. By 3-5 weeks the SV40-infected cultures appeared to be transformed, exhibiting the characteristics of continued division, decreased contact inhibition, decreased surface adhesion, and capacity to form clones at low density. The noninfected culture of each pair, however, manifested none of these qualities until 21/2-3 months following explantation. Two other criteria of transformation, ability to grow in suspension and formation of tumors in the host at the site of inoculation of cells, were not tested.

Chromosome Counts and FMF Patterns. In Table 1 are listed the cell lines studied and data characterizing their chromosome and DNA content and distributions. FMF patterns for some of these cultures are shown in Fig. 1. CAK is a murine cell line established several years ago in this laboratory (15) by spontaneous transformation of an embryonic culture. It has maintained a karyotype similar, but not identical, to its euploid origin and serves as a reference point for SV40 transformed mouse lines derived from similar stage mouse embryos.

The SV40-transformed murine cell lines are all heteroploid, in contrast to the spontaneously transformed CAK murine cell line. However, this is not a general rule since many, if not most, spontaneously transformed murine lines are heteroploid and the human SV40 transformed line (434SV) studied

Table 1. Coefficients of variation of chromosome number and G1 FMF peaks

Coefficient of variation Mean

chromosome Chromosome G 1 Cell line number number FMF peak

CAK 40.7 3.2 8 L1SVgl 73.5 6.8 13 L1SV92 73.4 4.1 7 L1SVr 72.7 7.6 13 434SV 46.7 3.7 - -


OAK

LISV(~I A YlSVgZ L

LISVd3

6 14

Fig. 1. FMF patterns of some of the cell lines used in this study. Channel numbers indicating relative DNA content are shown on absissa.

here is close to diploid in chromosome number . An impor tan t point which will be discussed la ter is the re la t ionship between the dis t r ibut ion of chromosome numbers within a cell line and the var iance of the G1 peak in the F M F

pa t te rn for tha t cell line. As can be seen there is a propor t ional re la t ionship between these two variables.

Sex Chromosome Replication in SV40-Transformed Lines. To deter- mine whether the la te repl icat ing pa t te rn of the Y and the inactive X chromosomes is re ta ined in SV40- t rans fo rmed cells, four mouse female lines, one mouse male line, and one human female line were pulse labeled with

Table 2. Percent unlabeled sex chromosomes in early-S pulsed cells

% of labeled Metaphases with metaphases with

Total labeled specific unlabeled evidence of a late Line metaphases chromosome replicating chromosome

1. L1SVgl 87 68 78.2 2. L1SV92 66 37 56.1 3. L1SV94 46 32 69.6 4. L1SV95 21 12 57.1 5. L1SVd3 42 22 52.4 6. 434SV 35 15 42.9


tritiated thymidine early in the S phase (11 h prior to harvesting). The results are shown in Table 2. Banding patterns of the cells revealed the unlabeled chromosome(s) to be an X or Y. Figures 2-4 are photographs of typical cells before and after autoradiography. A proportion of cells in these cultures were of high ploidy and retained several late replicating X chromosomes. Figure 2 is an example of one of these cells.

Line 434SV was not trypsin-banded, but in all cases the unlabeled chromosome was a submetacentric C-group chromosome consistent with the X. A representative 434SV cell is shown in Fig. 3.

Expression of X-Linked Genes. Line 434SV is heterozygous at two X-linked loci, PGK and G6PD. A clonal study of normal cells of this culture was reported earlier and showed that only two kinds of clones were recovered, Pgk' Gda and Pgk 2 Gd B, as expected if XCI is chromosomal. The SV40- transformed 434 culture was studied in the same way as untransformed 434 clones. The results are shown in Table 3 and, as can be seen, only two types of

Fig. 2. Autoradiogram of a L1SV21 cetl showing 3 unlabeled (arrows) chromosomes. Insert shows banded X chromosomes from the cell prior to autoradiography.


Fig. 3. Autoradiogram of a SV434 cell showing one unlabeled X chromosome.

clones were found, the same as was found for normal clones. Cells were also X-rayed with 176 or 352 rads and then cloned to see if any recombinants could be obtained. Again only the two parental clonal types were recovered. One interesting result involved X-raying and analysis of 55 clones from a previously isolated SV40 clone expressing Pgk 2 GdB; 53 of these clones were Pgk 2 Gd B and 2 were Pgk ~ Gd a.

DISCUSSION

There are three interesting findings in this study: (1) the relationship of chromosome number variation and FMF patterns; (2) sex chromosome replication patterns of SV40-transformed cells; and (3) X-linked gene expression in the double heterozygous human 434SV culture.

Chromosome Number Variation and FMF. In 1971 Kramer, Petersen, and Van Dilla (19) reported a lack of correlation between chromosome number variation and the variation in D N A content as determined by FMF.


Fig. 4. (A, top) Banded cell of L1SVd3; (B, bottom) autoradiogram of same L1SVd3 cell showing 2 unlabeled Y chromosomes.


Table 3. PGKandG6PDisozyme types of clones

PgkIGd A Pgk2Gd B PgklGd B PgkZGd A

Untreated 9 24 0 0 X-rayed 30 138 0 0

That is, even though some lines (e.g., HeLa) showed marked variability in chromosome number distribution, their FMF distribution (G1 peak) was no greater than lines with a narrow chromosome number variation (e.g., W1- 38). They hypothesized that the DNA in HeLa and other aneuploid lines exists as a continuous fiber during most of the cell cycle and is cut up in a variable fashion to generate the chromosomes seen at mitosis. In this way the lack of correlation between DNA variance at G1 and chromosome number variance at mitosis could be explained. A thorough test of this idea requires similar cell lines with the same mean chromosome number but different chromosome number distributions. From Table 1 it can be seen that three of our cell lines (L1SVQ1, L1SV92, and L1SV~3) are pertinent to this hypothesis; they have similar mean chromosome counts (-73), but their chromosome number distributions are different (L1SV~I and L1SV~3 have large CVs while L1SV~2 has a relatively low CV). The FMF CVs of these lines are proportional to their chromosome number distribution CVs. Furthermore the CAK line shows this same pattern (low chromosome number CV and low FMF CV). Thus, our data do not support the hypothesis of Kraemer et al.

Sex Chromosome Replication Patterns. Many neoplasms originating in human females have a significantly lower incidence of cells exhibiting a Barr body (20). Since the Barr body, as well as late replication, is a cytologic expression of the inactive X chromosome (21), this observation could indicate the presence of two functional X chromosomes in a single cell as a consequence of transformation. Alternatively, this could reflect loss of the inactive X from the aneuploid cells or interference with Barr body formation. Romeo and Migeon (11) reported that the incidence of sex chromatin decreased from 30% to 10% after SV40 transformation. The authors specu- late that this decrease could result from the early loss of the heterochromatic X chromosome.

In nontransformed female mouse cultures and a spontaneously transformed near-diploid mouse line (21, 22), approximately 55% and 71%, respectively, of metaphase cells labeled in early S Contain a chromosome of size and configuration consistent with the X (the inactive x) . In 4 heteroploid SV40-transformed female mouse lines a range of 57.1-78.2% of cells contained a late replicating X, identified by trypsin banding (Table 2). Many of these cells, in the hypotetraploid range, contained two late replicating X chromosomes, and it was possible to find very large cells that had retained more than 2 inactive X chromosomes. We have also shown that the delay in


replication initiation of the Y chromosome (21) is similarly conserved after SV40 transformation.

The difference between the end of replication is not as great in the mouse as it is in human cells. For this reason, the inactive X is difficult to detect in late-S pulsed metaphases. The mouse line L1SVQ1, however, was studied with a late-S pulse technique (4 h prior to harvest). Of 50 labeled metaphases, 4, or 8%, contained only one labeled chromosome, the same frequency as seen in nontransformed cultures (21). Thus our results indicate that there is no effect of SV40 cell transformation on replication patterns of sex chromosomes nor is there any increased tendency for inactive X chromosomes to be lost in these cultures.

Expression of X-Linked Genes. One can imagine several ways in which SV40 cell transformation might effect expression of X-linked genes. There could be a direct effect on the expression of the active allele, such as repression or modification. Repression of the active allele for any of the three loci (Gd, HGPRT, Pgk) studied in the work of Romeo and Migeon (11) and our own would have been detected. Since this was .not the case, we can exclude repression of active X-linked alleles-as a general effect of SV40 transformation. Modification as expressed by electrophoretic change could have been detected for the Gd and Pgk genes and again such effects were not detected. Another possible direct effect of SV40 transformation would be the reactivation of the inactive allele. This could lead to two active X-linked alleles in a cell, and in the case of the Gd locus would have been detected by the presence of a unique hybrid band upon electrophoresis. The hybrid band was not seen in either our extensive clonal studies or the work of Romeo and Migeon. Our studies should also have detected reactivation at the Pgk locus if it occurred. Thus it appears that there is no evidence for a direct effect of SV40 cell transformation on the Gd, Pgk, or HGPRT loci.

Another way in which SV40 transformation might affect X-linked gene expression is indirectly through chromosomal rearrangements (e.g., somatic crossing over) which could change the activation states of segments of the two X chromosomes. Proof of such an effect would be the appearance of a new phenotype in a double heterozygote. In this study the linkage relationships of the Gd and Pgk alleles were Pgk 2 Gd B and Pgk 1 Gd g and only those two cell phenotypes were found. A new phenotype, either Pgk ~ Gd B and/or Pgk 2 Gd h, would have been clearly detected, but was not in over 200 clones from both untreated and X-rayed SV40-transformed cultures. Romeo and Migeon analyzed 7 clones from a G d / H G P R T double heterozygote and also saw only parental phenotypes. In fact somatic crossing over has not been detected in any cell culture system (23). One would not expect new X chromosome activation states due to somatic recombination to occur with high frequency; so all we can conclude is that under our experimental condition (SV40 transformation and irradiation of up to 352 rads) such reactivation states


were not detected. It is also possible that somatic recombination could occur between X chromosomes but not lead to new activation states.

The two aberrant clones (Pgk ~ Gd A) that appeared from an X-rayed Pgk 2 Gd B clone could be explained in a trivial fashion, that is, that the original clone was not pure. However, several phenotypic assays of the parental clone revealed no evidence of Pgk 1 Gd A cells. I f these cases are examples of chromosomal reactivation, proof of such origin would be difficult. They could be recombinants or possibly represent loss of the active X chromosome with subsequent reactivation of the inactive X chromosome. A careful cytological analysis of such clones might provide some circumstantial evidence regarding this point (e.g., Angle X chromosome and /o r rearranged X chromosome), but none would likely be definitive. The results of these studies indicate that SV40 cell t ransformation has no detectable effect on either sex chromosome replication or X-linked gene expression.

A C K N O W L E D G M E N T S

Dr. Raskind was the recipient of an N I H fellowship through a Medical Scientist Training Program (GM 07266) during the course of this work. Dr. S.M. Gartler is a recipient of an N I H Research Career Award. The work was supported by N I H grant G M 1523. We gratefully acknowledge the expert technical assistance of Barbara Price, the help given us with the F M F work by Dr. J. Callis, and we are grateful to Dr. E. Giblett for performing the P G K

assays.

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x chromosome inactivation and sv40 transformation of mammalian cells

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