Lung cancer and the human gene for ribonucleotide reductase subunit
Diana M. Pitterle, Young-Chul Kim, Ethel M.C. Jolicoeur, Youjia Cao, Kathy C. O’Briant, Gerold Bepler
Department of Medicine, P.O. Box 2610, Duke University Medical Center, Durham, North Carolina 27710, USA
Received: 18 September 1998 / Accepted: 10 May 1999
Abstract. LOH11A is a region of Chromosome (Chr) 11p15.5
where 75% of lung cancers show loss of heterozygosity (LOH).
Clinical and cell biological studies suggest that LOH11A contains
a tumor/metastasis suppressor gene. We have mapped this region
(650 kb) using overlapping genomic P1/PAC/BAC clones, and one
of the genes that we have identified is RRM1. This gene encodes
the large subunit (M1) of ribonucleotide reductase, the heterodi-
meric enzyme that catalyzes the rate-limiting step in deoxyribo-
nucleotide synthesis. By comparing our genomic sequences with
the previously published cDNA, we have found that the human
gene is composed of 19 exons. It is oriented telomere to centro-
mere and is Alu rich. In order to verify that RRM1 maps within the
boundaries of LOH11A, we assessed the frequency of LOH at a
SacI polymorphism within intron IX of the gene. We observed
LOH in 48% (15/31) of informative lung tumor specimens. To
determine whether RRM1 was mutated in tumors, SSCP analysis
of the 19 RRM1 exons was performed. No mutations were re-
vealed in 12 pairs of normal and tumor DNA samples. Immuno-
blots on protein extracts from normal/tumor pairs indicated that a
protein of the expected size was present in both. Our conclusion is
that RRM1 lies within the LOH11A region, but that its exons are
not mutated in tumors. The potential for RRM1 to act as a tumor
suppressor is discussed.
Annually in the United States, lung cancers cause more deaths than
breast, colon, and prostate cancers combined (Parker et al. 1997).
Multiple mutations are involved in the development of lung can-
cers, and recent reviews have described some of the genes known
to be involved (Huebner et al. 1997; Kok et al. 1997; Pitterle et al.
1998). In addition, lung tumors frequently harbor DNA deletions
and/or rearrangements which can be detected as a loss of hetero-
zygosity (LOH). Chromosome 11p15.5 has at least two regions
that exhibit frequent LOH in lung tumors (O’Briant and Bepler
1997). The more centromeric region (LOH11A) is defined by the
polymorphic marker D11S12. LOH at D11S12 is observed in 88%
of small-cell carcinomas, 57% of squamous-cell carcinomas, and
40% of adenocarcinomas. In addition, this LOH is associated with
tumor stage and is observed more frequently in tumors that have
metastasized. These clinical data suggested that a gene in the vi-
cinity of D11S12 acts as a tumor/metastasis suppressor (Bepler et
Recently we developed a detailed map of Chr 11p15.5 cover-
ing the region from the hemoglobin (HBB) gene cluster through
marker D11S860, a span of over 1400 kb encompassing LOH11A
and D11S12 (Bepler et al. 1999). In the course of this mapping, our
work revealed that the RRM1 gene was within 50 kb of D11S12.
Its genomic structure and precise localization with respect to
D11S12 are reported here. Analysis of paired lung tumor and
normal DNA specimens verified that LOH is observed within this
gene. SSCP analysis of 19 exons of RRM1 in 12 sets of paired
samples strongly suggests that its exons are not mutated in these
tumors. Our results are discussed in light of recent studies that
show RRM1 has a malignancy suppressing affect (Fan et al. 1996,
Materials and methods
Isolation and analysis of genomic clones.
D11S12 was used as a
probe to isolate P1 phage genomic clone RMC11P010 (Berkeley Labora-
tories, Berkeley, Calif.). Sequences derived from the ends of RMC11P010
were used to isolate genomic clone 267B3 from a human PAC library
(Genome Systems, Inc., St. Louis, Mo.). EcoRI-digested fragments of the
human insert were shotgun-subcloned into pGEM-4Z and sequenced with
the SP6 and T7 primers using the fluorescent dideoxy-terminator method.
Internal primers (116) were designed as needed and obtained from Gibco-
BRL Custom Primers (Life Technologies, Inc., Gaithersburg, Md.). Most
sequences were run on an ABI Prism 377 DNA sequencer according to the
manufacturer’s instructions. In a few cases, dideoxy-terminator sequencing
was done manually with T7 Sequenase Version 2.0 (Amersham Life Sci-
ence, Cleveland, Ohio). In order to verify that subclones covering the entire
genomic fragment had been recovered, sequencing was also done directly
on PAC 267B3 with Big Dye chemistry from ABI. Four regions of the
PAC DNA were not recovered as EcoRI subclones and were obtained after
PCR amplification of the PAC DNA followed by direct sequencing of the
PCR product. Sequences were analyzed and aligned by use of the Wis-
consin Package for sequence analysis from the University of Wisconsin
Genetics Computer Group (575 Science Drive, Madison, Wis.) or the
program Sequencher, version 3.0, from Gene Codes Corporation (2901
Hubbard Street, Ann Arbor, Mich.).
Pulsed field gel analysis.
Genomic clones were digested with NotI,
BssHII, and MluI alone and in pairwise combinations, and the fragments
were separated on a 1% agarose gel overnight (16 h) with a Bio-Rad CHEF
DRIII System at the following settings: 2.6 s initial switch time, 6.6 s final
switch time, 6.0 V/cm, 120° included angle. Fragment sizes were estimated
in reference to bands in a 5-kb ladder (Bio-Rad, Hercules, Calif.). The
DNA bands were stained with ethidium bromide and digitally recorded
under UV illumination.
Fluorescence in situ hybridization.
Genomic DNA of P1 clone
RMC11P010 was prepared from overnight cultures with Plasmid Maxi
columns (Qiagen, Santa Clarita, Calif.). In order to generate a fluorescent
probe, the DNA was nick-translated in the presence of SpectrumOrange-
dUTP (Vysis, Downers Grove, Ill.). Similarly, a Chr 11 specific probe, the
alpha 11 centromeric satellite DNA, was labeled with fluorescein-
conjugated dUTP (SpectrumGreen, Vysis). Metaphase chromosomes were
prepared from normal human lymphocytes by standard procedures (Barch
Correspondence to: D.M. Pitterle
The nucleotide sequence data reported in this paper have been submitted to
GenBank and have been assigned the accession number AF107045.
Mammalian Genome 10, 916–922 (1999).
© Springer-Verlag New York Inc. 1999