[Cancer Research 59, 2038-2040, May 1, 1999]
© 1999 American Association for Cancer Research
[Cancer Research 59, 2038-2040, May 1, 1999]
© 1999 American Association for Cancer Research
Genetic and Functional Analyses Exclude Mortality Factor 4 (MORF4) as a Keratinocyte Senescence Gene1
Steven D. Bryce,
Nicholas R. Forsyth,
Sara A. Fitzsimmons,
Louise J. Clark,
Michael J. Bertram,
Andrew P. Cuthbert,
Robert F. Newbold,
Olivia M. Pereira-Smith and
E. Kenneth Parkinson2
Beatson Institute for Cancer Research, CRC Beatson Laboratories, Bearsden, Glasgow, G61 1BD United Kingdom [S. D. B., N. R. F., S. A. F., L. J. C., E. K. P.]; Roy M. and Phyllis Gough Huffington Center on Ageing, Baylor College of Medicine, Houston, Texas 77030 [M. J. B., O. M. P-S.]; and Departments of Biology and Biochemistry, Brunel University, Uxbridge, Middlesex, UB8 3PH United Kingdom [A. P. C., R. F. N.]
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ABSTRACT
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Approximately 50% of immortal human keratinocyte lines show loss of heterozygosity of chromosome region 4q33-q34, and the reintroduction of chromosome 4 into one such line, BICR 6, causes proliferation arrest and features of replicative senescence. Recently, a candidate gene, mortality factor 4 (MORF4), was identified in this region and sequenced in 21 immortal keratinocyte lines. There were no mutations or deletions, and two of the seven lines that showed loss of heterozygosity at 4q33-q34 were heterozygous for MORF4 itself. Furthermore, the transfer of a chromosomal segment containing the entire MORF4 gene did not mimic the senescence effect of chromosome 4 in BICR 6. These results suggest that the inactivation of MORF4 is not required for human keratinocyte immortality.
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Introduction
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The immortalization of human keratinocytes is thought to be a permissive condition for the rapid progression of human squamous carcinoma cells (1)
and is associated with p53 dysfunction (1
, 2)
, homozygous deletion of the INK4A locus (2)
, p16INK4A dysfunction (2)
, and high levels of telomerase (2)
. In addition, immortal human keratinocytes derived from SCC-HN3
show frequent LOH on chromosome 4 at D4S408/D4S1535 in the 4q33-q34 region (2)
, and similar regions of loss have been reported in carcinoma of the bladder (3)
, esophagus (4)
, and head and neck (5)
in vivo. Therefore, a gene in this chromosomal region may also be a bone fide tumor suppresser in vivo. Chromosome 4 causes features of replicative senescence when introduced into several human cell lines (6)
, including an SCC-HN line, BICR 6, with LOH on 4q.4
Recently a candidate gene, MORF4, a truncated variant of a novel family of putative transcription factors, was reported to map to 4q33-q34.1 and to mimic the senescence effect of chromosome 4 (7)
. Because this gene mapped close to one of the minimally deleted regions in our panel of cell lines, we tested whether MORF4 was a likely candidate for the keratinocyte senescence gene. The results show that MORF4 is unlikely to be this gene.
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Materials and Methods
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Cell Lines and Culture Methods.
The human keratinocyte lines used in this study are listed in Table 1
and were cultured in DMEM containing 20 mM HEPES, antibiotics, 10% v/v fetal bovine serum, and 0.4 µg/ml hydrocortisone (Sigma, Poole, Dorset, United Kingdom ). The human fibroblasts were cultured in the same medium with 20% v/v fetal bovine serum and without hydrocortisone.
Microcell-mediated Chromosome Transfer.
The generation of the neomycin resistance-tagged human chromosome 4 fragment, F4, and the methods for transferring chromosomes into keratinocytes by microcell-mediated chromosome transfer have been described previously (7
, 8)
.
Measurement of Senescence and Population Doublings.
The number of population doublings was measured by recording the cell inputs and cell yields at each passage and using the formula mean population doublings = 3.32 (log10 cell yield - log10 cell input).
The BICR 6 cells that had received an intact copy of chromosome F4 were considered to be immortal when they had completed 50 population doublings because senescence was induced by the intact chromosome 4 within 20 population doublings (6)
.
Extraction of DNA from Cultured Cells.
DNA was extracted from 1 x 106-1 x 107 cells using QIAamp tissue kit (QIAgen Ltd) according to the manufacturer’s "blood and body fluid" protocol.
MORF4 Primer Design, PCR, and Sequencing.
Primers were designed such that the 3' ends of each oligonucleotide sequence hybridized to positions at which MORF4 differed from the other MRG family sequences (7)
, this ensured specific amplification and sequencing of MORF4. PCR was performed using the primers G1-F: 5'-AAATGGGCTA AATGCCGTAG-3' and G1-R: 5' -CACTTTACAG CATATCCCTG-3', in reactions containing final concentrations of 1 µM each oligonucleotide, 200 µ M each dATP, dCTP, dGTP, and TTP, 1.5 mM MgCl2, 1x Reaction buffer II (Perkin-Elmer), 1 unit of Taq polymerase (Perkin-Elmer AmpliTaq), and 100 ng of genomic DNA. Amplification, after a denaturation step for 3 min. at 94°C, used 30 cycles of 30 s at 94°C, 30 s at 60°C, and 1 min at 72° C, followed by a final extension step of 7 min at 72°C (Perkin-Elmer 9600 thermal cycler). Before sequencing, PCR products were purified using a QIAquick PCR purification kit (QIAgen Ltd.) following the manufacturer’s protocol.
MORF4-G1 PCR amplified products were sequenced using six internal primers (three acting in the forward direction and three in the reverse), designed using the same criteria as the PCR primers; S1-F: 5'-CAGTGTGCTG TATTCAGGAG-3', S2-F: 5' -ATTGGGATGA TGGGGTTCCG-3', S3-R: 5'-CAGACCAGAT GTCTTCTTTC C-3', S4-F: 5'-GCTCTTCTAT CTTCCTGCCG-3', S5-R: 5'-CCCAACATTA GGTTGAAGTA TT-3', and S6-R: 5' -CTAACCTCTT CAAAGCACAT CG-3'. Sequencing was performed using 3.2 pmol primer oligonucleotide, 40–80 ng of purified PCR product, and BigDye Terminator cycle sequencing reagents (Perkin-Elmer, Applied Biosystems). Reactions were conducted on a thermal cycler (PCT-100, Genetic Research Instrumentation Ltd.) using 25 cycles of 10 s at 95°C, 5 s at 55°C, and 4 min at 60°C and then run on an ABI 373 sequencer (Applied Biosystems).
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Results and Discussion
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We have designed MORF4-specific PCR primers and established that they are chromosome 4-specific by using a panel of mouse A9 cells, each carrying a single human chromosome. In particular, the primers do not amplify from other human chromosomes to which other members of the MRG family of sequences (7)
are reported to map (Fig. 1)
. We have sequenced, with specific internal primers, a MORF4 PCR product amplified from genomic DNA that was isolated from normal fibroblasts from many of the SCC-HN patients and one control sample. We also sequenced MORF4 in 21 immortal keratinocyte lines mainly derived from human SCC-HN (see Table 1
). We found no mutations or deletions. Seven of these lines—BICR 6, BICR 18, BICR 19, BICR 22, BICR 31, BICR 78 and BICR 82—show LOH at D4S408/D4S1535, close to the MORF4 locus at 4q33-q34.1. Any base differences from the NheI fragment of MORF4, which induces senescence in other cell lines after transfection (7)
, were not cancer-specific and are, therefore, polymorphisms. Examples of these polymorphisms are shown in Fig. 2a
. The fact that MORF4 is so polymorphic yields valuable information because, often, different MORF4 alleles can be distinguished on the basis of sequence. The high frequency of polymorphisms also stresses the need to sequence normal material from the same donor when looking for cancer-specific mutations of this gene. Two lines of the seven that showed LOH at the MORF4 region, BICR19 and BICR22, were in fact heterozygous for MORF4 itself; that is, MORF4 is not in the minimal LOH interval. We were able to show this because of the highly polymorphic nature of MORF4 (compare BICR19 with BICR16 and BICR31 in Fig. 2b
). This result also argues against the hypothesis that the loss of one wild-type MORF4 allele can contribute to immortality through haplo-insufficiency (see Ref. 9
). As a direct test of the candidacy of MORF4, we introduced a fragment of human chromosome 4, F4 (7)
, into a keratinocyte line, BICR 6, that is known to senesce after the introduction of an intact copy of human chromosome 4.4 Three colonies were isolated after G418 selection, and none of them senesced, whereas 11 of 15 of the control HeLa colonies did senesce as reported previously (7)
. All of the three BICR 6-F4 clones retained the exogenous MORF4 gene as determined by the polymorphisms at positions 49, 115, 615, 730, and 865 of MORF4 after sequencing. The sequence of one clone is shown in Fig. 2c
. The entire sequence was carefully examined for interstitial deletions and point mutations of MORF4 in these three clones, and none were seen. Even a 1-bp deletion of one MORF4 allele would be easily recognized as a second sequence on the trace. The retention of the introduced MORF4 gene at such a high frequency in immortal segregants would not be expected if MORF4 were the keratinocyte senescence gene on chromosome 4 (10
, 11)
.
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Fig. 1. Amplification of a specific 1526-bp MORF4 product by PCR only from genomic DNA containing the relevant portion of human chromosome 4. Lanes 1–7 contain human chromosomes 1, 4, 5, 7, 11, 15, and X, respectively, on an A9 murine background; Lane 8 contains total human genomic DNA; Lane 9 contains a chromosome 4 fragment (containing MORF4; see Ref. 7
) on an A9 murine background; Lane 10 contains A9 murine genomic DNA,; Lane 11 contains no DNA (negative control); Lane 12 contains a 1-kb DNA ladder (Life Technologies, Inc.)
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Fig. 2. Sequence traces at polymorphic loci in the MORF4 gene, bases (arrows) are numbered such that position +1 corresponds to the first base after the 5' flanking repeat in the MORF4 sequence (7)
. Bases are labeled R when both A and G peaks are present, Y when C and T peaks are present, and W when A and T peaks are present. a, heterozygous sequence showing multiple polymorphic loci in normal fibroblast (from the same patient from which the BICR 31 SCC-HN line was derived) DNA. b, sequences homozygous for different sequence alleles of MORF4 (BICR 31 and BICR 16) and a sequence heterozygous for MORF4 (BICR 19) in SCC-HN tumor-derived cell lines. c, sequence of exogenous MORF4-containing chromosome fragment F4, endogenous MORF4 from BICR6 and a BICR 6-chromosome F4 hybrid (F4.02) showing retention of the exogenous MORF4 allele.
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Our previous work (2
, 6
, 7
.4)
has indicated that there may be a novel senescence gene on human chromosome 4, and the work of others (3, 4, 5)
suggests that the same chromosomal region contains a genetic suppressor of certain human carcinomas. The evidence presented above suggests that MORF4 is unlikely to be either the gene that causes senescence in SCC-HN cells or the suppresser of SCC-HN tumors in vivothat maps to the same chromosomal region (5)
. The keratinocyte senescence gene could still map to 4q33-q34, but other regions of 4q also display high frequencies of LOH both in SCC-HN lines (2)
and in in vivo tumors (5)
. In particular, the 4q24-q26 region has been implicated in the pathogenesis of SCC-HN (5)
and in keratinocyte immortalization (2)
, but current evidence suggests that this region carries a gene that suppresses tumor development without causing replicative senescence (12)
. Further work is, therefore, required to map and identify the chromosome 4 gene involved in keratinocyte senescence and immortalization.
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ACKNOWLEDGMENTS
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We thank Professor John Wyke for critical review of the article and the Cancer Research Campaign and the Association for International Cancer Research for the financial support of the work.
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FOOTNOTES
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1 Supported by grants to E. K. P. and R. F. N. from the Cancer Research Campaign and the Association for International Cancer Research and by NIH Grants R37AGO5333 (to O. M. P-S.) and T32AG00183 and F32AGO5732 (to M. J. B.). 
2 To whom requests for reprints should be addressed, at the Beatson Institute for Cancer Research, CRC Beatson Laboratories, Garscube Estate, Switchback Road, Bearsden, Glasgow, G61 1BD United Kingdom. Phone: 44-141-330-3653; Fax: 44-141-942-6521; E-mail: ekp1n{at}udcf.ac.gla.uk
3 The abbreviations used are: SCC-HN, squamous cell carcinoma of the head and neck; MORF4, mortality factor 4; LOH, loss of heterozygosity; MRG, MORF4 related gene.
4 N. R. Forsyth, S. A. Fitzsimmons, A. Cuthbert, R. F. Newbold, S. D. Bryce, and E. K. Parkinson, unpublished data.
Received 1/11/99.
Accepted 3/19/99.
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REFERENCES
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Edington K. G., Loughran O., Berry I. J., Parkinson E. K. Cellular immortality : a late event in the progression of human squamous cell carcinoma of the head and neck associated with p53 alteration and a high frequency of allele loss. Mol. Carcinog., 13: 254-265, 1995.[Medline]
-
Loughran O., Clark L. J., Bond J., Baker A., Berry I. J., Edington K. G., Ly I-S., Simmons R., Haw R., Black D. M., Newbold R. F., Parkinson E. K. Evidence for the inactivation of multiple replicative lifespan genes in immortal human squamous cell carcinoma keratinocytes. Oncogene, 14: 1955-1964, 1997.[Medline]
-
Polascik T. J., Cairns P., Chang Y. H., Schoenberg M. P., Sidransky D. Distinct regions of allele loss on chromosome 4 in human primary bladder cancer. Cancer Res., 55: 5396-5399, 1995.[Abstract/Free Full Text]
-
Hammond Z. T., Kaleem Z., Cooper J. D., Sundareson S., Patterson G. A., Goodfellow P. J. Allelotype analysis of esophageal adenocarcinomas : evidence for the involvement of sequences on the long arm of chromosome 4. Cancer Res., 56: 4499-4502, 1996.[Abstract/Free Full Text]
-
Perhouse M. A., El-Naggar A. K., Hurr K., Lin H., Yung W. K. A., Steck P. A. Deletion mapping of chromosome 4 in head and neck squamous cell carcinoma. Oncogene, 14: 369-373, 1997.[Medline]
-
Ning Y., Weber J. L., Killary A. M., Ledbetter D. H., Smith J. R., Pereira-Smith O. M. Genetic analysis of indefinite division in human cells : evidence for a cell senescence-related gene(s) on human chromosome 4. Proc. Natl. Acad. Sci. USA, 88: 5635-5639, 1991.[Abstract/Free Full Text]
-
Bertram M., Berube N. G., Hang-Swanson X., Ran Q., Leung J. K., Bryce S., Spurgers K., Bick R. J., Baldini A., Ning Y., Clark L. J., Parkinson E. K., Barrett J. C., Smith J. R., Pereira-Smith O. M. Identification of a gene that reverses the immortal phenotype of a subset of cells and is a member of a novel family of transcription factor-like genes. Mol. Cell. Biol., 19: 1479-1485, 1999.[Abstract/Free Full Text]
-
Cuthbert A. P., Trott D. A., Ekong R. M., Jezzard S., England N. L., Themis M., Todd C. M., Newbold R. F. Construction and characterization of a stable human : rodent monochromosome hybrid panel for genetic complementation and genome mapping studies. Cytogenet. Cell Genet., 71: 68-76, 1995.[Medline]
-
Fero M. L., Randel E., Gurley K. E., Roberts J. M., Kemp C. J. The murine gene p27Kip1 is haplo-insufficient for tumour suppression. Nature (Lond.), 396: 177-180,
-
England N. L., Cuthbert A. P., Trott D. A., Jezzard S., Nobori T., Carson D. A., Newbold R. F. Identification of human tumour suppressor genes by monochromosome transfer : rapid growth-arrest response mapped to 9p21 is mediated solely by the cyclin D-dependent kinase inhibitor gene, CDKN2A (p16INK4A). Carcinogenesis (Lond.), 17: 1567-1575, 1996.[Abstract/Free Full Text]
-
Robertson G. P., Furnari J. W., Lugo T. G., Huang H-J. S., Cavanee W. K. In vitro loss of heterozygosity targets the PTEN/MMAC 1 gene in melanoma. Proc. Natl. Acad. Sci. USA, 95: 9418-9423, 1998.[Abstract/Free Full Text]
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Pershouse M. A., Ligon A. H., Pereira-Smith O. M., Killary A. M., Yung W. K. A., Steck P. A. Suppression of transformed phenotype and tumorigenicity after transfer of chromosome 4 into U251 human glioma cells. Genes Chromosomes Cancer, 20: 260-267, 1997.[Medline]
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ABSTRACT
Introduction
