Download Escape from Senescence in Hybrid Cell Clones Involves Deletions

(CANCER RESEARCH56. 241-245. January 15. 1996]
Advances
in Brief
Escape from Senescence in Hybrid Cell Clones Involves Deletions of Two Regions
Located on Human Chromosome lq'
Christina Karlsson,2 GöranStenman, Patrick J. Vojta, Erik Bongcam-Rudloff,
and Ylva Paulsson
J. Carl Barrett, Bengt Westermark,
Department of Pathology. University Hospital. Dag Hammarskjdlds v. 20, S-751 85, Uppsala [C. K., Y. P., E. B-R., B. WI; Department of Cell Biology, Faculty of Health
Sciences. Linkoping Unisersitv. S-541 85, Linkoping 1G. SI, Sweden; Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, Research
Triangle Park. North Carolina 27709 fP. J. V., J. C. B.J: and Curriculum in Genetics and Molecular Biology, The University of North Carolina at Chapel Hill, Chapel Hill,
North (‘arolina27599 [P. J. V.. J. C. B.J
brids betweenyoung and senescentcells, DNA synthesisis inhibited
Abstract
in the nucleus of the young cell. Treatment of senescent cells with
Human normalcellshavebeenshownto undergoa limitednumberof
inhibitors of protein synthesis before fusion abrogates the inhibition,
cell doublings,a phenomenontermedcellularsenescence.
Human chro
indicating that the process is mediated by certain proteins (5). Further
mosome1 hasbeenimplicatedin thisprocess,
andseverallinesof evidence
indicate
that
there
is a senescence-inducing
evidencein supportof this ideacomesfrom experimentsdemonstrat
gene or genes on human
chromosomelq. Our approach to analyzethe senescence-inducing
effect
of chromosome1 includesthe useof somaticcell hybrid revertants.We
show here that fusion of a hypoxanthinephosphoribosyltransferase
negativemousecellline(A9) containinga humanneo-tagged
chromosome
1 with an immortal hamster cell line (1OW-2)results in cell hybrids that
senesceafter a few population doublings. Rare revertants that had es
caped senescence were obtained after one large fusion experiment. Thirty
five nonsenescent
hybrids
were obtained
from
a total of approximately
hybrids
tions of chromosome 1 were observed in any of the hybrids. Deletion
mappingrevealedthat 11 (56%) of the hybridsanalyzedhad lostoneor
more markerson chromosomelq. Two regionswith deletionswere de
tected,oneof whichhasbeenshownto be implicatedin the senescence
inducing effect exerted by chromosome 1 following monochromosome
transfer(P. J. Vojta el aL, manuscriptsubmittedfor publication).The
present study suggeststhat two separateloci on human chromosomelq
may be of importancefor the inductionof senescence.
Moreover,thisset
of nonsenescent
revertantscouldbe usefulfor futuredetailedanalysesof
the senescence-inducing
loci.
Introduction
Normal human fibroblasts in culture have a finite life span, and the
cells cease to divide after a limited number of population doublings.
This phenomenon, known as cellular senescence,was first described
by Hayflick and Moorhead (1). It is characterized by the progressive
loss of proliferating cells until the entire population has lost its
mitogenic potential. A link between age-related functional decline in
vivo and senescence in vitro is suggested by the clear relationship
between the maximum life span of diverse animal species and the
replicative life span of their cultured fibroblasts (2). Furthermore,
there is an inverse relationship between the maximum population
doubling capacity of cells in culture and the donor age (3).
Cell fusionexperimentshaveshownthathybridsof normal(mortal)
and immortal cells have a limited growth potential (4). Thus, the
mortal phenotype is dominant over the immortal phenotype. In hy
Received 10/5/95; accepted 11/28/95.
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.
work
was
supported
by the
Swedish
Cancer
Society,
Magn.
Bergvalls
2 To
whom
requests
of chromosome
1 suppressed
the infinite
growth
potential
of
a Syrian hamster cell line (1OW-2; Ref. 7), a human uterine endome
trial carcinoma cell line (8), human neuroblastoma cells (15), and cell
Smith and Smith (16) and Hensler et al. (17). The region on human
chromosome 1 responsible for the induction of senescencehas been
mapped to 1q23—lqter(7), but the exact localization on chromosome
1 is not known. The aim of the present investigation was to further
analyze
the
SI@ effect
of human
chromosome
1 in immortal
Syrian
hamster cells (1OW-2) by generating somatic cell hybrid revertants
after fusion with mouseA9 cells, carrying a normal humanchromo
some 1 (A9—!neo). To increase the revertant frequency and possibly
inactivatethe SI locus by insertionalmutagenesis,the A9—lneo cells
were infected with a murine retrovirus (MoMuLV) before cell fusion.
Twenty-five cell hybrids that had escaped senescence were isolated
and analyzed with regard to chromosome numbers, presenceof human
chromosome1, and loss of chromosome1 loci.
Materials and Methods
Cell Culture Conditions. The immortal Syrian hamstercell line lOW-2
was chosen because it has a near-diploid
stable karyotype
(Table
1), is
nontumorigenic,and undergoessenescence
after receivinga single copy of
human chromosome 1 (7, 18). The mouse A9—l neo cells are immortal and
contain a single humanchromosome1 (Table 1) taggedwith a neomycin
resistance
California,
marker (kindly provided by Dr. Eric Stanbridge, University
of
Irvine, CA). This cell line is HPRT-negative
and thus unable to
grow in HAT medium.All cells were grown in DMEM supplementedwith
10% FCS (GIBCO)
and antibiotics.
MoMuLV Infection. SparselyseededmouseA9—lneo cells were incu
batedovernightin M0MuLV-containingmedium,harvestedfrom virus-pro
ducingcells (kindly providedby Dr. BjOrnVennstrOm,KarolinskaInstitute,
Stockholm,Sweden),and supplementedwith polybrene(8 @g/ml).
The cells
werethenwashedandpreparedfor fusion experiments.
Founda
lion, and Pharmacia AB.
46-18-55 89 31.
Transfer of specific chromosomes using the microcell fusion tech
nique has revealed that certain human chromosomes, viz. X, 1,4,6,7,
9, 11, and 18 induce cellular senescencein certain cell lines (7—14).
lines assignedto senescencecomplementationgroup C by Pereira
was confirmed by fluorescencein situ hybridization analysis using a
chromosome1-specificpainting probe. No visible translocationsor dele
I This
competenthumanfibroblasts(6).
Transfer
1
million hybrids,and 25 of theseweresubjectedto further analysis.The
presence of a single copy of human chromosome 1 in the revertant
ing an inhibition of DNA synthesis after microinjection of polyade
nylated RNA from senescent human fibroblasts into proliferation
3 The
abbreviations
used
are:
SI,
senescence
inducing;
HPRT,
hypoxanthine
phospho
ribosyl transferase; FISH, fluorescence in situ hybridization, HAT, hypoxanthine amin
for
reprints
should
be addressed.
Phone:
46-18-66
38
27;
Fax:
opterinethymidine,MoMuLV, Moloney murineleukemiavirus; STS,sequence-tagged
site.
241
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1996 American Association for Cancer Research.
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Fig. 1. Hybrid cells after fusion of A9—lneo cells to lOW-2. A, hybrids that senescedafter fusion. B, after different periods of time, the senescentcells loosened abruptly from the
dishes and died. C, example of a hybrid clone that did not senesce,round/small morphology. D, example of a hybrid clone that did not senesce,elongated morphology.
Cell Fusionof 1OW-2andA9—1
neoCells. Exponentially
growing,virus
infected,A9—lneocells,and 10W-2 cells weretrypsinizedandsuspendedin
were as recommended
by the manufacturer.
Chromosomes
were counter
for 1 mm. Then, 10 ml
stainedwith propidiumiodide. Fluorescencemicroscopywasperformedin a
Leica confocalmicroscope,and the resulting imageswere processedin the
LeicasoftwareprogramTCS,transferredto a Macintoshcomputer,andfurther
processedin AdobePhotoshop.
PCR. To detectpossibledeletionsof chromosome
1 sequences
in thecell
Eagle's MEM containing 25% PEG-l000 were added, and the mixture was
hybrids, STS analysis was performed. Chromosomal DNA was prepared
Eagle's MEM supplemented with 10% newborn calf serum. Equal numbers of
lOW-2 and A9—lneo cells were mixed and divided into five samples.The
cells werethensuspendedin 10ml Eagle'sMEM containing35%polyethyl
ene glycol MW
1000 (PEG-l000)
and 7% DMSO
incubatedfor another2 mm. The sampleswere diluted with 40 ml Eagle's
MEM, and finally 40 ml Eagle'sMEM supplementedwith 10%normalcalf
serumwere added.The experimentwas performedat 37°C.The cells were
platedsparselyin 10-cmdishes,andafter4 h, the mediumwassupplemented
with HAT (100p@M
hypoxanthine,0.8 @LM
aminopterine,and16,@M
thymidine)
and G418 (800 ,.@g/m1).Control fusion experiments were perfonned using A9
accordingto standardproceduresand subjectedto PCR usingeight different
chromosome1-specificmarkers.Two markers(D1S450andDJS200)mapto
lp andsix markers(NCF2,D1S212,D1S412,D1S505,HLXJ, andDISJO2)to
lq. NCF2 andHLXI aretranscribedgenesequencesthat havebeenlocalized
to 1q25 (19)
and lq4l—42
(Ref.
20; Humane
Genome
Database),
respectively.
The exact physical positions of the remaining markers are not known. The
cells containinga humanchromosome8 taggedwith a neomycinresistance
marker(A9—8
neo;kindly providedby Dr. StefanImreh,KarolinskaInstitute) tentative order of the eight markers is lpter-D1S450-DJS200-cen-NCF2/
D1S212-D1S412-D1S505/HLXJ-DJSJO2-lqter
and lOW-2 cells (I X 106 cells of each cell type).
Cytogenetic and FISH Analyses. Chromosomepreparationswere made
from exponentiallygrowingcultures.CellswereharvestedafterColcemid
exposurefollowedby hypotonictreatmentandfixation in methanol:acetic
acid.
Slides were subsequently G banded or routine stained with Giemsa. A total of
20—25metaphaseswas analyzedper cell clone. The presenceof human
chromosomeI in the hybrid cloneswasconfirmedby G bandingand/orFISH
analysisusing a humanchromosome1-specificpainting probe(WCP1; Im
agenetics,Framingham,MA). The probe was labeledwith SpectrumGreen
fluorophore. The conditions for hybridization
and posthybndization
for NCF2 and HLXI
(21,
22).@ Primer
sequences
were chosen from intronic and 3' untranslated regions to
enhance the probability of human-specific amplification on mouse and hamster
backgrounds.Primer sequenceswere 5'-GACATFACAG'lTFGGAAGT
GU-3' and 5'-CTfGGGCCTGGACTl@-GGGTG-3'for NCF2. 11W se
quences were 5'-GCCTACCTCCCG-ACATFCACG-3' and 5'-GGGCIC
GAGTCAGAGAAACAGT-3'. PCR was performed in 20-pi reactions
4 P. J. voju,
p@ A.
Futreal,
L.
A.
Annab,
H.
Kato,
0.
M.
Pereira-Smith,
Barrett,manuscriptsubmittedfor publication.
washes
242
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and
J. C.
EFFECT OF HUMAN CHROMOSOME I IN CELL FUSION HYBRIDS
containing200 ng DNA, 1.0 @M
primers,200 @m
deoxynucleotidetriphos
phates,1X PCR buffer, and 0.5 units Amplitaq polymerase(Perkin Elmer/
Cetus) and 1.5 mMMgC12for the HLXJ reaction and 1.0 nmiMgC12for NCF2.
Reactionconditionswere 1 min/94°C
for 1 cycle followed by 36 cyclesof 1
min/94°C,1 minI58°C,
and 1 minI72°C
for HLXJ, using the Perkin Elmer/
Cetus480 format, and 30 s/94°C
for 1 cycle followed by 36 cycles of 15
s/94°C, 30 s/58°C, and 30 sfl2°C for NCF2,
A
@—
using the Perkin Eliner/Cetus
ci
9600 thermocycler format. All other markers were detected using primer
sequences and conditions based on the Human Genome Database entries.
Thesereactionswererun usingstandardconditionsfor 9600formatreactions,
essentiallyas describedabovefor NCF2. MgC12andannealingtemperatures
were adjustedfrom 0.9 to 1.75mM and 55 to 59°C,respectively,to obtain
human-specificamplificationof thesemarkersonrodentgenomebackgrounds.
PCRproductswereanalyzedon 1.5or 2% agarosegels.
I
Results
Presentation of the Experimental Approach. Our approach was
to analyze the SI effect of human chromosome 1 by the use of somatic
cell hybrids. A9—l neo cells containing a human chromosome 1
taggedwith a neomycin resistancemarker were infected with Mo
MuLV. Senescence was induced by fusion of HPRT A9—1neo
mouse cells containing human chromosome 1 with the immortal
Syrian hamstercell line lOW-2 (HPRT@).Hybrid cells wereisolated
by selection in G4l8 and HAT-supplemented medium. Escape from
senescencemay be caused by deletions, inactivating mutations, or
translocationsin the SI locus. Retroviral infection could also lead to
integration of the virus into the SI locus.
Whole Cell Fusions. Pilot experiments were performed in which
106 uninfected A9—l neo cells were fused to an equal number of
1OW-2 cells. After approximately 1 week of growth in selection
medium, cells started to die and resistant clones were emerging. After
another 2 weeks, the clones ceased to grow and showed a senescent
phenotype with large cells with an abundant cytoplasm (data not
shown).
When
a similar
number
of mouse
cells
containing
a neo
tagged human chromosome 8 were fused to 1OW-2cells, the resulting
Table 1 Chromosomeconstitution
and in vitro growth morpholog
andNo.
immortalized hybrid clonesy
ofparental
of chromosomesPresence
humanCell
of
lineMean
morphologyParental
Range
chromosome1Cell
lineA9I neo52.0
small10
W-244.
irregularHybrid
I
48—56
44—45Large,
+Round,
cell
clone565.0
+Elongated869.3
63—88
+Elongated2774.0
+ND2864.4
65—83
65—87
+ND2961.0
54—75
+Elongated3078.2
like31ND
+ND3288.9
small36116.1
+Elongated37122.6
+ND39126.1
like4199.2
58—74
67—90
ND
62—104
107—128
104-137
118—138
+ND4386.6
90-107
+Elongated4489.4
+ND5061.1
+ND5168.2
+ND5271.7
+Elongated6388.5
+Elongated6976.7
+ND7069.3
+ND7179.9
+ND7282.5
+Elongated7478.1
75—98
81-99
59—77
60—80
62—100
75—109
67—88
61—82
71—95
67—88
74—87
65—90
small8074.8
+Elongateda
ND,
56—75
+ND―II72.4
not
done.
+10
W-2
+Round,
+lOW-2
signal).A, A9-l ncocells containingthe mousechromosomesand humanchromosome
1. B, a hybrid clone containingan apparentlynormal human chromosomeI.
+Round,
hybrids did not show signs of senescence(data not shown). We found
that the frequency of hybrid formation in the A9—lneo X 1OW-2cell
fusion experiments was approximately 1 hybrid/103 cells of each
parent cell line.
To get a sufficient number of revertants, iO@MoMuLV-infected
A9—lneo cells were fused to an equal number of lOW-2 cells. After
a few weeks, resistant clones were obtained. Most hybrid clones
243
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EFFECT OF HUMAN CHROMOSOME
I IN CELL FUSION HYBRIDS
A
@
B
marker Iiow@@Ja Iii
J28 1311321361 37f 411
@@I@F52I @I
Fig. 3. A, deletion mapping of the immortal
%ioss
izedhybridsusingSTSanalysis.Horizontalaxis,
parentalcell lines(lOW-2 andA9—lneo)andthe
13
hybrids that contained deletions on human chro
mosome 1. D15200 and D15450 are located on
Ip and the other markers on lq. B, idiogram of
4
35
human chromosome I showing two regions
30
(DR-l and DR-2) in which common deletions
were detected in the revertant hybrids.
17
q25
I DR-I
q41-42
I
I absent
@+/LIIpresent
1
started to senesceafter 2—3
weeks (Fig. 1A). Cell clones that did not
clones went into crisis (Fig. 1B). In the second selection, 27 cell
PCR and Southern blot. However, no such sequenceswere found (data
not shown). It is therefore unlikely that the loss of the SI gene(s) was
causedby insertional mutagenesisby the retrovirus.
Deletion Mapping of the Immortalized Cell Hybrids. To inves
ligate whether the escape from senescencewas due to deletions of
cloneswere isolated.After 3 months,35 of 53 cell cloneswere still
chromosome1 sequences
in anyof thehybrids,DNA wasisolatedfrom
actively proliferating and considered to have escaped senescence.
Thus, of approximately 106 hybrids, 35 hybrids became revertants,
representing a revertant frequency of 3.5 X l0@. Twenty-five rever
tant hybrids were subjected to further analysis. The hybrids could be
23 clones, and STS content analysis was performed using eight different
chromosome 1 markers. As shown in Fig. 3A, two common regions of
deletions were found on the q arm. In 9 (39%) of 23 hybrids, the NCF2
marker, mapped to 1q25 was lost. Three of these hybrids had also lost
divided into threegroupsregardingtheir morphology,i.e., elongated,
D15212,
round/small (A9 morphology), and large/irregular (1OW-2 morphol
ogy). Fig. 1, C and D, exemplify the round/small and the elongated
phenotypes, respectively.
Cytogenetic and Molecular Genetic Analyses. The immortal
phenotype of the A9—lneo/1OW-2 hybrids could be due to inactiva
tion of the SI locus on human chromosome 1. This inactivation could
either be the effect of mutations, deletions, or rearrangements of
chromosome 1 sequences or caused by retroviral integration. We
analyzed the revertant hybrids (passages 8—15)with regard to the
presenceof human chromosome 1 and possible gross rearrangements
involving this chromosome using cytogenetics and/or FISH. Hybrid
ization with a chromosome 1-specific painting probe revealed the
presence of one copy of human chromosome I in all 25 hybrids
analyzed (Table 1). Examples of hybridized metaphasesare shown in
Fig. 2, A (A9—lneo parent cell line) and B (hybrid clone 44). No gross
rearrangements of chromosome 1, i.e., translocations, insertions, or
and HLXJ, located at 1q41—42,
were found in 8 (35%) and 7 (30%)
show any typical signs of senescencewere isolated and serially
propagated. In the first selection, 26 cell clones were picked, and of
these,10 startedto show signs of senescence,and after a while the
deletions,were observedin any of the cell lines. The total chromo
some number (mean and range) in each hybrid cell line was also
determined by counting the chromosomes in 20—25cells from each
hybrid clone using cells from passages8—15
(Table 1). About 66% of
the hybrids had a mean chromosome number of <85 chromosomes,
2 1% had 85—100
chromosomes, and about 13% had a mean value of
> 100 chromosomes.
There
was no correlation
between
the morphology
of thecell clonesandchromosomenumbers.All of thecell hybridswere
analyzed with respect to the presenceof MoMuLV proviral DNA by
which maps immediately
distal to NCF2. Deletions
of D1S505
hybrids,respectively.DJSIO2,a markerdistal to HLXJ was lost in 4
(17%) hybrids. Deletions in both ofthese regionshadoccurredin 7(30%)
of the hybrids. Fig. 3B shows the two lq regions (designated DR-l and
DR-2) in which common deletions were detected.
Discussion
Previous studies have shown that human chromosome I induces
senescence in the immortal Syrian hamster cell line 1OW-2 (7). In
the present study, we transferred chromosome I to 1OW-2 cells by
fusing the cells to mouse A9 cells carrying one copy of chromo
some 1. Becausethe vast majority of clones that emerged were mortal,
we have drawn the conclusion that the presenceof mousechromosomes,
donated by the A9—lneo cells, does not interfere with the SI process.
Moreover, control experiments showed that A9 cells canying human
chromosome 8 were incapable of inducing senescence when fused with
lOW-2 cells. The presentstudy thereforestrengthensthe notion that
chromosomeI containsoneor moreSI loci.
Many of the hybrid cell clones were found to undergo several cell
doublings and did not senesceuntil they had formed small, discrete
colonies. A few clones could even be serially propagated before
senescence.Thus, the introduction of chromosome 1 did not lead to an
immediategrowth inhibition; rather the presenceof chromosome1
apparentlyinitiated a programof senescence with a delayed onset,
244
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EFFECT OF HUMAN CHROMOSOME
which became evident as cessation of growth after several rounds of
division. The process thereby mimics the aging of normal cells, and
the finding therefore strongly argues that chromosome 1 harbors
genes involved in cellular senescence.
In the present study, we attempted to facilitate the identification and
cloning of putative SI genes by using retrovirus-mediated mutagene
sis. The advantage with retrovirus-mediated mutagenesis is that the
proviral DNA not only causes a mutation but also tags the DNA
region of interest, which facilitates mapping and cloning of the rele
vant gene. Surprisingly, we detected no proviral MoMuLV DNA in
the hybrids either by Southern blotting or PCR. Although we were
able to produce a rather large number of revertants by our experimen
tal protocol, it is unfortunate that we obtained no detectable retrovirus
tags to facilitate further analyses.
Loss of heterozygosity in the long arm of chromosome 1 has been
detected in several human tumors, including breast, gastric, ovarian,
basal cell, and non-small cell lung carcinoma (23—26),indicating the
presence of tumor suppressor genes on lq. In a recent study, Hensler
et a!. (17) found that lq induces a senescent phenotype when trans
ferred into three different cell lines belonging to complementation
group C, as defined by Pereira-Smith and Smith (16). The present
study suggests that the long arm of chromosome 1 also induces
senescencein A9—lneo X 1OW-2fusion hybrids.
I IN CELL FUSION HYBRIDS
3. Schneider, E. L., and Mitsui, Y. The relationship between in vitro cellular aging and
in vivo human age. Proc. Nati. Acad. Sci. USA, 73: 3584—3588, 1976.
4. Bunn, C. L., and Tarrant, G. M. Limited lifespan in somatic cell hybrids and cybrids.
Exp. Cell Res., 127: 385—396,
1980.
5. Pereira-Smith, 0. M., Fisher, S. F., and Smith, J. R. Senescent and quiescent cell
inhibitors of DNA synthesis:membrane-associated
proteins.Exp. Cell Res., 160:
297—306,1985.
6. Lumpkin, C. K., McClung, J. K., Jr., Pereira-Smith,0. M., and Smith, J. R. Existence
of high abundanceantiproliferativemRNA's in senescenthumandiploid fibroblasts.
Science(WashingtonDC), 232: 393—395.
1986.
7. Sugawara, 0., Osbimura, M., Koi, M., Annab, L. A., and Barreu, J. C. Induction of
cellular senescencein immortalized cells by human chromosome I . Science (Wash
ington DC), 247: 707—710,1990.
8. Yamada,H., Wake, N., Fujimoto, S., Barren, J. C., and Oshimura,M. Multiple
chromosomescarryingtumor suppressoractivity for a uterineendometrialcarcinoma
cell line identified by microcell-mediated chromosome transfer. Oncogene, 5: 1141—
1147, 1990.
9. Ning, Y., Weder, J. L., Killary, A. M., Ledbetter, D. H., Smith, J. R., and Pereira
Smith, 0. M. Genetic analysis of indefmite division in human cells: evidence for a
cell senescence-relatedgene(s) on human chromosome 4. Proc. Natl. Aced. Sci. USA,
88: 5635—5639,1991.
10. Klein, C. B., Conway, K., Wang, X. W., Bhamra, R. K., Lin, X., Cohen, M. D.,
Annab, L., Barrett, J. C., and Costa, M. Senescenceof nickel-transformed cells by an
x chromosome:
possible
epigenetic
control.
Science
(Washington
DC),251:796—
799, 1991.
11. Ogata, T., Ayusawa, D., Namba, M., Takahashi, E., Oshimura, M., and Oishi, M.
Chromosome 7 suppressesindefinite division of nontumorigenic immortalized
human fibroblast cell lines KMST-6 and SUSM-1. Mol. Cell. Biol., 13: 6036—
6043, 1993.
12. Sandhu, A. K., Hubbard, K., Kaur, G. P., Tha, K. K., Ozer, H. L., and Athwal, R. S.
Senescenceof immortal humanfibroblastsby the introduction of normal human
chromosome6. Proc.Nail. Aced. Sci. USA, 91: 5498—5502,
1994.
The SI locus has previously been mapped to 1q23—lqterby Sug
awara et a!. (7). These data are consistent with our findings and
further suggestthat two regionson lq are responsiblefor the induc
tion of senesence: one located at 1q25 (region DR-i including NCF2)
and the other at lq4l—42 (region DR-2, including HLXJ).
The finding of a high frequency of concomitant deletions of two
regions on iq suggeststhat at leasttwo SI loci may have to be deletedto
allow for infinite growth. Even if this argumentholds true, the finding of
revertants without any detectable deletion, or deletions in only one
region, may be expected;useof infrequently spacedmarkersfor deletion
analysis is a relatively insensitive method and does not reveal small
deletions or point mutations. Thus, in hybrids where no deletions were
found, the escapefrom senescencemay be explained by the fact that the
deletions were two small to detect. Another possibility is that one or more
hamster genes,of importance for the induction of the complete senes
cence program. have been inactivated in theseparticular hybrids.
The cloning and characterization of SI genes will increase the
understandingof the mechanismsbehind the loss of limited growth
potential in neoplastic cells. It is likely that one or several senescence
genesare lost in the majority of immortal tumor cells. This panelof
nonsenescenthybrids may provide a tool for future work to identify
13. Gualandi, F., Morelli, C., Pavan, J. V., Rimessi, P., Sensi, A., Bonfatti, A., Gruppioni,
R., Possati,L., Stanbridge,E. J., and Barbanti-Brodano,G. Induction of senescence
and control of tumorigenicity in BK virus transformed mouse cells by human
chromosome 6. Genes Chromosomes & Cancer, 10: 77-84, 1994.
14. Sasaki, M., Honda, T., Yamada. H., Wake, N., Barrett, J. C., and Oshimura, M.
Evidence for multiple pathways to cellular senescence.Cancer Res., 54: 6090—6093,
1994.
15. Bader, S. A., Fasching, C., Brodeur, G. M., and Stanbridge, E. J. Dissociation of
suppressionof tumorigenicity and differentiation in vitro effected by transfer of single
humanchromosomesinto neuroblastomacells. Cell Growth & Differ., 2: 245—255,
1991.
16. Pereira-Smith, 0. M., and Smith, J. R. Genetic analysis of indefinite division in
human cells: identificationof four complementationgroups.Proc. Natl. Acad. Sci.
USA, 85: 6042—6046,1988.
17. Hensler,P. J., Annab, L. A., Barrett.,J. C., and Pereira-Smith,0. M. A geneinvolved
in control of human cellular senescenceon human chromosome lq. Mol. Cell. Biol.,
14: 2291—2297,1994.
18. Oshimura, T., Hesterberg, J., and Barrett, I. C. An early, nonrandom karyotypic
change in immortal Syrian hamster cell lines transformed by asbestos: trisomy of
chromosome11.CancerGenet.,22: 225—228,
1986.
19. Francke, U., Hsieh, C-H., Foellmer, B. E., Lomax, K. J., Malech, H. L. and Leto, T.
Genes for two autosomal recessive forms of chronic granulomatous disease assigned
to 1q25 (NCF2) and 7q1 1. 23 (NCFI). Am. J. Hum. Genet., 47: 483—492,1990.
20. Nishimura, D. Y., Purchio, A. F., and Murray, J. C. Linkage localization of TGFB2
andthehumanhomeobox
geneHLX1to chromosome
lq. Genomics,
15:357—364,
1993.
21. Dracopoli, N. C., Bruns, G. A. P., Brodeur, G. M., Landes, G. M., Matise, T. C.,
Seldin, M. F., Vance, J. M., and Weith, A. Report of the first internationalworkshop
the senescencegene(s) located on human chromosome lq.
on humanchromosome1mapping1994.Cytogenet.Cell Genet.,67: 143—174,
1994.
Acknowledgments
We thank Meta Lindstrdm and Asa Johanssonfor skillful technical
assistance.
22. Murray, J. C., Buetow, K. H., Weber, J. L, Ludwigsen, S., Scherpbier-Heddema, T.,
and Manion, F. A comprehensivehuman linkage map with centimorgandensity.
Science (Washington DC), 265: 2049—2073,1994.
23. Chen, L-C., Dollbaum, C., and Smith, H. S. Loss of heterozygosity on chromosome
lq in human breastcancer.Proc. NatI. Acad. Sci. USA, 86: 7204—7207,1989.
24. Sano, T., Tsujino, T., Yoshida, K., Nakayama, H., Haruma, K., Ito, H., Nakamura, Y.,
Kajiyama,G., andTahara,E. Frequentlossofheterozygosityonchromosomeslq. Sq.
andl7p in human gastric carcinomas. Cancer Res., 51: 2926—2932,1991.
25. Bare, J. W., Lebo, R. V., and Epstein, E. H., Jr. Loss of heterozygosity at chromosome
1q22 in basal cell carcinomas and exclusion of the basal cell nevus syndrome gene
References
1. Hayflick, L., and Moorhead, P. S. The serial cultivation of human diploid cell strains.
Exp. Cell Res., 25: 585—621,1961.
2. Rohme, D. Evidencefor a relationshipbetweenlongevity of mammalianspeciesand
life-spans of normal fibroblasts in vitro and erythrocytes in vivo. Proc. Natl. Acad.
Sci.USA, 78:5009—5013,
1981.
from this site.CancerRes.,52: 1494—1498,
1992.
26. Tsuchiya, E., Nakamura, Y., Weng, S.-Y., Nakagava, K., Tsuchiya, S., Sugano, H.,
and Kitagawa,T. Allelotype of non-smallcell lung carcinoma—comparison
between
loss of heterozygosity in squamouscell carcinoma and adenocarcinoma. Cancer Res.,
52: 2478—2481,1992.
245
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Escape from Senescence in Hybrid Cell Clones Involves
Deletions of Two Regions Located on Human Chromosome 1q
Christina Karlsson, Göran Stenman, Patrick J. Vojta, et al.
Cancer Res 1996;56:241-245.
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