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[Cancer Research 62, 2498-2502, May 1, 2002]
© 2002 American Association for Cancer Research

Advances in Brief

Deficiency of Retinoblastoma Gene in Mouse Embryonic Stem Cells Leads to Genetic Instability1

Lei Zheng, Andrea Flesken-Nikitin, Phang-Lang Chen and Wen-Hwa Lee2

Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78245


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Genetic instability has been recognized as a hallmark of human cancers. Retinoblastoma (Rb) tumor suppressor protein has an essential role in modulating cell cycle progression. However, there is no direct evidence supporting its role in maintaining genetic stability. Here, we developed a sensitive method to examine the level of chromosome instability by using retrovirus carrying both positive and negative selectable markers that integrated randomly into individual chromosomes, and the frequency of loss of this selectable chromosomal marker (LOM) in normal mammalian cells was measured. Our results showed that normal mouse embryonic stem (ES) cells had a very low frequency of LOMs, which was less than 10-8/cell/generation. In Rb-/- mouse ES cells, the frequency was increased to approximately 10-5/cell/generation, whereas in Rb+/- ES cells, the frequency was approximately 10-7/cell/generation. LOMs was mediated mainly through chromosomal mechanisms and not through point mutations. These results, therefore, revealed that Rb, with a haploinsufficiency, plays a critical role in the maintenance of chromosome stability. The mystery of why Rb heterozygous carriers have early-onset tumor formation with high penetrance can be, at least, partially explained by this novel activity.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
Genetic instability is one of the most important hallmarks of cancer (1) . Associations of tumor suppressors with the process of chromosome behavior provide possible links between carcinogenesis and genetic instability. Aneuploidy, chromosome structural rearrangements, centrosome amplification, and gene amplification have been observed in p53-deficient cells (2) and, more recently, in APC-deficient cells (3) , which supports a potential role of tumor suppressors in the maintenance of genetic stability.

The roles of Rb3 in cell cycle regulation and differentiation are well established and can explain how Rb suppresses tumor growth, but do not completely explain why cancer susceptibility results from loss of Rb function (4) . In particular, the mystery as to why the inactivation of Rb leads to multiple genetic alterations that predispose cells to the process of tumorigenesis remains unsolved. In addition to its role in G1 progression, several lines of evidence suggest that Rb also plays a significant role at G2-M phases. First, the hypophosphorylated form of Rb, the functional form, is present in these cell cycle stages. It appears that Rb becomes dephosphorylated as Rb interacts with protein phosphatase 1{alpha} specifically during G2-M phases (5) . The yeast homologue of protein phosphatase 1{alpha} has been shown to be essential for kinetochore function, execution of mitotic kinetochore/spindle checkpoint, and faithful chromosome segregation (6) . A regulatory role in the activity of protein phosphatase 1{alpha} mediated by Rb would be consistent with other potential activities of Rb in G2-M progression. Second, phosphorylation of Rb in G1-S phases has a coordinated effect on mitotic cyclin induction and degradation in G2-M phases (7) . Third, overexpression of Rb in S phase arrests cells at G2 phase (8) . Fourth, when treated with microtubule-destabilizing agents, cells lacking functional Rb do not finish mitosis properly but exit M phase and undergo a new cycle of DNA replication, leading to hyperploidy (9) . Finally, p53-mediated G2-M arrest in response to DNA damage requires the presence of functional Rb (10) . Taken together, these results suggest that mitotic division cannot be completed in a controlled manner without functional Rb.

Rb may also directly modulate chromosome segregation. Rb has been shown to associate with Hec1, a conserved regulator of multiple mitotic events (11 , 12) . Hec1 interacts with the SMC family of proteins, which are chromosome structural proteins essential for establishment of sister chromatid cohesion and chromosome condensation (13) . Inactivation of Hec1 in either mammalian cells or yeast cells, either by microinjection of specific anti-Hec1 antibodies or by introducing genetic mutations, leads to severe chromosome missegregation resulting in lethality (11 , 14) . This essential function of Hec1 appears to be mediated in part by its interaction with the SMC family of protein (14) . The interaction between Rb and Hec1 occurs specifically in G2-M phase (12) . It has been demonstrated that the fidelity of chromosome segregation in yeast cells is enhanced on induced expression of exogenous human Rb, and this activity requires the specific interaction between Rb and Hec1 (12) . However, the role of Rb for maintaining genetic stability in mammalian cells remains to be shown.

Here, we developed a sensitive method for examining the level of chromosome instability in normal mammalian cells by using a retroviral system to integrate randomly a selectable marker on individual chromosomes. The frequency of LOMs was measured, and our results showed increased frequency of LOMs (to ~10-5/cell/generation) in mouse Rb-/- ES cells compared with that in Rb+/+ cells. The frequency of LOMs in Rb+/- cells was also moderately increased. Further analysis indicated that the loss of the fusion genes is most likely caused by gross chromosomal changes.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Isolation, Culture, and Genotyping of Mouse ES Cells.
Rb +/+, Rb+/- and Rb-/- ES cells were isolated from blastocysts of the same litter of mouse embryos taken from pregnant intercrossed Rb+/- mice (15) following a previously described procedure (16) . Briefly, blastocysts were harvested from pregnant mice on embryonic day 3.5 and cultured on a feeder layer derived from mouse embryonic fibroblasts. The embryos were allowed to hatch for 4–6 days and the inner cell mass was moved away from the trophoblast cells, disaggregated, and cultured on the feeder layer in the medium containing DMEM, 15% fetal bovine serum, and leukemia- inhibitory factors. The pluripotential individual ES colonies were picked, transferred onto a freshly prepared feeder layer, and amplified. To determine the genotype of the ES cells, genomic DNA was extracted, and PCR was performed as described previously (17) . The selected three genotypes of the ES cells were karyotyped and had apparently normal chromosome pattern.

Selection of Individual ES Clones Infected with HygTK Retrovirus.
The HygTK fusion gene (18) was inserted between the two LTRs derived from Moloney murine leukemia virus in a previously described vector (19) . The resultant vector pLHL1-HygTK was transfected into the retroviral packaging cell lines and the HygTK virus was harvested. Approximately 2 x 106 ES cells, isolated as above, described at the 12th passage with the genotype of Rb+/+, Rb+/-, and Rb-/-, respectively, were grown on a hygromycin-resistant feeder layer derived from embryonic fibroblasts of transgenic mice expressing the hygromycin phosphotransferase gene (The Jackson Laboratory). Cells were infected by the HygTK virus for 24 h in the presence of Polybrene and subsequently treated with 250 µg/ml hygromycin after another 24 h. Hygromycin-resistant clones were picked after 9 days of selection and afterward maintained in the ES cell culture medium containing 200 µg/ml hygromycin. Clones were individually amplified, and PCR analysis indicated that all of the hygromycin-resistant colonies carried the integration of the HygTK fusion gene (data not shown).


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
Establishing a Method for Measuring the Globally Genetic Instability in Mammalian Cell.
To follow the dynamic changes of individual chromosomes, a selectable marker was permanently integrated on the chromosome by retrovirus-mediated gene transfer, which would be expected to harbor a single copy of this marker randomly on individual chromosomes (19) . By tracing this marker in multiple virally infected clones, one would have a global view of genetic alterations on chromosomes in general rather than at one specific locus. For the feasibility of detecting the presence or absence of the marker, we chose a fusion gene that combines the hygromycin phosphotransferase (Hyg) gene and herpes simplex virus type 1 thymidine kinase gene (TK) in frame. Translation of this fusion gene into a single bifunctional enzyme protein (designated hereafter as HygTK) confers both resistance to hygromycin and sensitivity to ganciclovir (18) , which provides both positive and negative selectivity.

The HygTK fusion gene was inserted between two LTRs derived from Moloney murine leukemia virus (19) ; and thereby, its expression was under the control of the LTR promoter (Fig. 1A)Citation . In an attempt to explore the overall frequencies of LOMs regardless of the viral integration loci, more than 50 individual hygromycin-resistant colonies with approximately equal number of cells were mixed together. The mixture of cells was maintained in the hygromycin-containing medium, and then transferred to a selection-free medium in which the cells were to be propagated for about 10 generations. Before this propagation step, 1 x 107 cells were removed from the hygromycin-containing medium and immediately seeded in the ganciclovircontaining medium, and the ganciclovir-resistant colonies were counted to obtain Eo (Fig. 1B)Citation . After the propagation (in the selection-free medium), the cell number was counted; the number of generations through which the cells had been propagated was calculated (usually between 9 and 10 generations); 1 x 107 cells were seeded in the ganciclovir-containing medium; and, after 12 days of ganciclovir-selection, the ganciclovir-resistant colonies were counted to obtain Ep (Fig. 1B)Citation .



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  		Fig. 1. A method for measuring the frequency of LOMs using the retroviral-integrated HygTK fusion gene as reporter. A, schematic structure of the retrovirus carried the HygTK fusion gene. B, experimental steps outlined for measuring the frequency of loss of heterozygosity. C, formula for calculating the frequency of loss of heterozygosity. FeqLOM is the frequency of loss of the marker per cell per generation. Ep and Eo are designated as in B. n, the number of generations.

 
The ganciclovir-resistant colonies that arose were from those cells that had lost the functional TK gene and, thus, were no longer sensitive to ganciclovir. Maintaining the cells in the hygromycin-containing medium would have prevented the loss of the entire HygTK gene; therefore, ganciclovir-resistant cells were not detected or were detected at a frequency less than 10-8. The appearance of ganciclovir-resistant cells before the cells were propagated in nonselective medium would be expected, because the accumulation of mutations during this long-term culture could have inactivated the TK gene in some hygromycin-resistant cells. This frequency, however, was subtracted from that of ganciclovir-resistant cells analyzed after the propagation, and the resultant subtraction represented the frequency of cells that had the TK gene inactivated during the propagation. Finally, this subtraction was sequentially divided by the total cell number and by the generation numbers to deduce the frequency of LOMs at the HygTK-integrated loci in one cell per division generation (Fig. 1C)Citation .

Loss of Rb Gene Increases Frequency of LOMs.
The above method is ideal for cells with high colony forming efficiency. Normal human fibroblasts and mouse embryonic fibroblasts have poor colony-forming efficiency, at about 10-3 to 10-4. Therefore, it requires a substantial amount of cells for measuring LOMs by this method. Any immortalized cell lines, including cancer cell lines, are prone to genetic changes. The selection process in this procedure may create a bias toward certain changes and generate artifacts for this measuring. To circumvent these difficulties, we used ES cells to perform this experiment because of their normalcy and high colony-forming efficiency. Three mouse ES cell lines with normal karyotype from the same litter were used; one with the wild-type Rb, the other with one allele of Rb mutated at exon 20, and another one with both alleles mutated at exon 20 (15) . Our results indicated that the LOM frequency of Rb+/+ cells was lower than 10-8, the frequency of Rb+/- cells was between 10-7 and 10-6, and the frequency of Rb-/- cells was higher than 10-5 (Fig. 2)Citation . These results, therefore, suggest that the frequency of LOMs is increased in cells homozygous for the null mutation of Rb and moderately increased in cells heterozygous for Rb.



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  		Fig. 2. Increased frequency of LOMs in Rb-deficient ES cells. Three representative dishes selected for ganciclovir-resistant colonies are shown. A mixture of more than 50 individual hygromycin-resistant clones of ES cells infected with the HygTK retrovirus was propagated about 10 generations without any selection. About 107 cells of each genotype were seeded on 10-cm dishes and selected for ganciclovir-resistant colonies. Pictures were taken after 12 days of selection. The frequencies of LOMs were calculated based on the results collected from multiple dishes

 
To determine whether the experiments with mixed viral-infected colonies would reveal the average frequencies of LOMs regardless of the viral integration loci, we examined the LOM frequencies at individual integration loci. Viral-infected cells were selected by hygromycin and hygromycin-resistant clones were randomly picked and individually cultured. PCR analysis indicated that all of the selected clones carried the integration of the HygTK fusion gene (as shown later in Fig. 4Citation ). The ES cells carrying the HygTK fusion gene were maintained in hygromycin-containing medium for at least 6 days before LOM frequency was analyzed following the same procedures for the experiments with the mixed clones.



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  		Fig. 4. Genotyping PCR of the HygTK fusion gene and the Rb allele. A, schematic diagram of the genomic structure of the HygTK-integrated loci. PCR primers for amplifying various regions of the HygTK gene are indicated at their relative positions. B, genotyping PCR of the HygTK gene. Lanes 1–5, representatives of the hygromycin-resistant clones that carry the HygTK fusion gene; Lanes 6–15, representatives of ganciclovir-resistant clones. The approximate size of PCR products and corresponding primers used are indicated. Bottom panel, two pairs of primers as indicated were used in the same reaction. Primers amplifying a region of the Brca1 genome were used as control. C, genotyping PCR showed that the heterozygosity of the Rb allele was retained. Lanes 1–8, representatives of hygromycin-resistant clones of Rb+/- ES cells that carry the HygTK fusion gene; Lanes 9–27, representative of ganciclovir resistant clones of Rb+/- ES cells that have lost the HygTK fusion gene. 236 bp, PCR products derived from the target allele; 151 bp, products derived from the wild-type Rb allele.

 
More than 20 individual clones of each genotype were analyzed to cover approximately one haploid number of chromosomes. The results indicated that individually viral-infected clones, although derived from the same parental cells, have different frequency of LOMs at the HygTK loci. A similar frequency of LOMs was observed with the same clone in repeated experiments. Considering that retroviral infection tends to integrate the HygTK marker in different chromosomal loci, we inferred from these results that different chromosomal loci vary in the frequency of LOMs. We summarized the number of clones with the frequency of LOMs that fell in the same logarithm range (Fig. 3A)Citation . Results from both sources of Rb+/+ ES cells were combined and showed that ~90% of the Rb+/+ clones had a frequency of LOMs lower than 10-8, and the rest of the clones had a frequency of LOMs between 10-8 and 10-6 (Fig. 3B)Citation . However, 80–90% of the Rb-/- clones had a frequency of LOMs higher than 10-6, and a number of clones (~30%) had a frequency of LOMs higher than 10-4. Karyotyping of these clones that had a frequency of LOMs higher than 10-3 revealed that they had an aneuploidy tendency (~10% of the cell population). Interestingly, although none of the Rb+/- clones had this LOM frequency higher than 10-4, ~60% of these clones had a LOM frequency between 10-7 and 10-5. These results suggested again that Rb-deficient cells have a significantly higher frequency of LOMs compared with that of wild-type cells. As shown in Fig. 3BCitation , the majority of clones had a frequency of LOMs similar to that of the mixed clones (Fig. 2)Citation , which suggests that the assay with the mixed clones was able to reveal an overall frequency of LOMs that closely represented the stability of the whole genome. It should be noted that the frequency determined by this procedure could be overestimated because the mutated cells may continue to propagate in nonselection medium. However, the relative frequencies of these three different genotype cells are consistent, regardless of the different procedures.



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  		Fig. 3. Frequencies of LOMs in individual infected clones. A, number of clones of each genotype of ES cells is listed in the table (A) with frequencies of LOMs falling in each indicated logarithm range. B, histogram shows the percentage of clones falling in each indicated logarithm range deduced from A.

 
Loss of the Marker Involves Larger Deletion of Chromosomal Event Instead of Point Mutation.
To explore the mechanisms underlying the loss of functional TK gene, we examined the status of the HygTK fusion gene in the ganciclovir-resistant cells by PCR. As shown in Fig. 4, A and BCitation , PCR was not able to amplify any fragments of the HygTK fusion from genomic DNA of the ganciclovir-resistant clones that were randomly picked. This result indicated that ~90% of ganciclovir-resistant clones had lost the entire HygTK fusion gene physically. It also suggested that point mutations or small deletions that could have inactivated the TK gene occurred at a low frequency (~10%). As suggested previously, potential mechanisms attributed to the loss of a whole gene include chromosomal loss, mitotic recombination, and interchromosomal rearrangement, which are all chromosome mechanisms (20) . Therefore, increased frequency of LOMs observed in Rb-/- cells suggests that Rb-deficiency would lead to chromosome instability.

Interestingly, retaining one wild-type Rb allele does not appear to be sufficient for the maintenance of chromosome stability, because Rb+/- cells also have a moderately increased frequency of LOMs compared with the wild-type cells. It would, however, be possible that chromosome instability is a consequence of the loss of the remaining wild-type Rb allele. To examine this possibility, these ganciclovir-resistant clones derived from Rb+/- cells were genotyped for the wild-type Rb allele by PCR. The result indicated the clones picked randomly all retained one wild-type Rb allele (Fig. 4C)Citation , which suggested the haploinsufficiency of Rb in maintaining chromosome stability.


   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
Genetic instability in Rb-deficient cells could be responsible for further genetic alterations involved in cancer development. Germ-line mutations in one Rb allele lead to the development of retinoblastoma in human and pituitary tumors in mice at very early ages and with nearly complete penetrance (15 , 21) . The remaining wild-type allele is lost as a somatic event and, as suggested, mainly through chromosome mechanisms (20 , 22) . It has been estimated previously, based on the mean number of tumors occurring in carriers of Rb, that the mutation rates in both events are nearly equal (21) .

In this study, by directly accessing the mutation rate, one view suggested that the mutation rate increases when the first Rb allele is inactivated. Chromosome instability in Rb-heterozygous cells could explain the high penetrance of tumor development with loss of the remaining wild-type allele. Nonetheless, the loss of the second Rb allele appears to be the threshold event in tumor development, given that it results in much more severe genetic instability, which might account for all of the following genetic alterations essential for tumorigenesis.

The possibility cannot be excluded that Rb could also suppress the frequency of point mutations or small deletions, considering that the HygTK gene may not serve as an appropriate reporter for small nucleotide changes. However, our observations implicating a low frequency of point mutations or small deletions in Rb-deficient cells is consistent with previous reports indicating that the loss of the remaining Rb allele in the majority of Rbs is mediated by chromosome mechanisms (20 , 22) . In addition, chromosome mechanisms appear to be the major cause of loss of heterozygosity during tumorigenesis based on studies with different tumor suppressor genes (23) .

In previous studies, reporters such as the adenine phosphoribosyltransferase (APRT) gene at a specific chromosome locus have been used to evaluate the frequency of LOMs (23 , 24) . The new method used in this study, by using retroviral infection to integrate the reporter randomly on different chromosomes, allows us to access comprehensively all of the chromosome behaviors in a cell. In addition, the loss of reporter is prevented by positive selectivity conferred by the same reporter gene; thus, the new method is able to evaluate precisely the level of chromosome instability.

Chromosome abnormality in Rb-deficient fibroblasts has not yet been reported. The average frequency of LOMs observed in Rb-/- cells is approximately 10-5. Classical methods such as multiplex fluorescent in situ hybridization and spectral karyotyping (25) would not be able to catch any aberrance if vast numbers of cells were not subjected to such analysis. By contrast, the new method appears to be more sensitive in evaluating the chromosome instability. However, unlike fluorescent in situ hybridization or spectral karyotyping, this new method does not reveal the types of chromosome aberrance directly. Our method has to be followed by traditional polymorphism marker analysis on the viral-integrated chromosome if one is interested in what types of chromosome mechanisms are involved.

Although nondisjunction is apparently a result of improper chromosome segregation, how other types of chromosome aberrance occur remains to be clarified. Chromosome mechanisms underlying LOMs in Rb-deficient cells are most likely comprised of multiple types of chromosome aberrance because Rb appears to be capable of modulating chromosome metabolisms from different, but intimately related, aspects including chromosome replication, segregation, and structural maintenance (4) . Given that Rb is regarded as the prototype for tumor suppressors, it will be even more interesting to examine the role of other tumor suppressors in maintaining chromosome stability by this new method. It is conceivable that the other tumor suppressors have a common role in the maintenance of chromosome stability, and such a role may be pivotal for their functions in tumor suppression.


   ACKNOWLEDGMENTS
 
We thank Dr. Stanley Cohen (Stanford University) for his generosity in providing pLLTX plasmid and Dr. Nicholas Ting for his critical reading of the manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by NIH Grants EY 05758 and CA58318. Back

2 To whom requests for reprints should be addressed, at Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, Texas 78245. Phone: (210) 567-7351; Fax: (210) 567-7377; E-mail: leew{at}uthscsa.edu Back

3 The abbreviations used are: Rb, retinoblastoma; LOM, loss of chromosomal marker; ES, embryonic stem; HygTK, fusion gene (or virus) of the hygromycin phosphotransferase gene and thymidine kinase; SMC, structural maintenance of chromosomes; LTR, long terminal repeat. Back

Received 12/ 6/01. Accepted 3/13/02.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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