- Email: [email protected]
The reactivation of telomerase activity in cancer progression
DEBATE
qke reactivation of telomerase activity in cancer progression JERRY W. SHAYAND WOODRING E. WRIGHT [email protected] [email protected] THEUNIVEtL~;ITYOFTEX.~.SSOVI'~'F.SlXRXMEDIC.~CENTF.LDEPART?,IEN'OF f CELLBIOLOGYA~XtNEUROSCIENCE,5323 HAaR'¢HINESBOULEVARD, DaLt~S,TX 75235-9039, USA.
There is increasing evidence suptx~rting the hypothesis that the shortening of telomeres is the clock that times cellular senescencet-7~ Most human primary tumors contain telomerase (the enzyme activity that prevents telomere shortening), while most normal tissues and cells lack this activity (Ref. 8 and reviewed in Refs 9,10). These observations contribute to the concept that normal cells lack sufficient proliferative capacity to undergo the repetitive cycles of mutation and clonal expansion that would be required to accumulate the multiple genetic abnormalities present in cancer cells. In most cases, escape from the proliferative limitations of cellular senescence by telomerase reactivation might be a prerequisite for the development of a malignant tumor cell. Although we believe that this scenario has a great deal of experimental support and is likely to represent real biology, there are a variety of subtleties that need to be considered. One of these, raised by Mel Greaves in the preceding article, which we will consider below, is that telomerase-positive cancer cells arise from tdomerase-positive stem cells with out the need for a reactivation step.
How does telomerase regulate telomere length? Telomere length is likely to be a good indicator of the proliferative history of telomerase-negative cells, in which telomeres shorten progressively with each :,ucceeding cell division 1-'~. However, telomere length might not be informative in telomerase-pcsitive cells. The 'appropriate' level of telomerase activity in any cancer cell should maintain a stable telomere length, regardless of whether the telomeres are 2 or 20kb long. Increased or decreased functional telomevase activity should result in a changing telomere length. At present, virtuaily nothing is known about the mechanism by which cells expressing telomerase regulate the length of their
telomeres n. The presence of shoa telomeres in many tumors 12-14 suggests that considerable telomere shortening occurred before the telomerase was reactivated, but clearly does not prove it, because telomere shortening could have continued in the presence of telomerase. By the same token, tumors with long telomeres need not be derived from stem cells that never repressed telomerase and, thus, never shortened their telomeres, because the telomeres could have been elongated after telomerase reactiw~tion. In both cases, the result miglt reflect a regulated process rather than a dysfunctional enzyme, because an appropriate ~ctivity must be obtained if a stabh~ telomere length is ultimately to be n:aintained. Telomere length in telomerase-positive tumor cells is, thus. not an effective discriminator about whether or not the cancer progenitor cell was a telomerase-positive stem cell. Regulation of telomerase activity The evaluation of telomerase is further complicated by the fact that telomerase-positive cells become telomerase-silem when they become quiescent (S. Holt et al., unpublisbed). This is not surprising, because an extraordinarily tight regulation of intracellular activity would be required to prevent telomere elongation in a telomerase-competent stem cell or germ cell that remained quiescent for many years. This observation has a variety of implications. On the one hand, it suggests that the most primitive stem cells that rarely divide might be protected from the effects of any anti-telomerase cancer therapies. On the other hand, it implies that evidence for the lack of telomerase achvi W in normal or tumor cells needs to be carefully interpreted. Normal diploid cells dividing in culture 15--E7 and hyperproliferative benign lesions, such as leiomyomass, do no: exhibit TIG APRIL 1996 VOL. 12 NO. 4
C~)pjxight©19%Et~'vlcrScicno:lld. Allnght.,reservcd. Ol(~9525/96/$15.00
129
telome~ose activity, so tim lack of telomerase in these situations clearly does not reflect the lack of mitotic activity. However, it is possible that the lack of detectable telomerase activity in normal tissues and some benign or premalignant lesions could reflect the lack of cell division, rather than the lack of telomerase-competent cells. Because telomerase activi W is detected in all phases of the cell cycle in immortalized cells and is only absent in GO (S. Holt et al., unpublished), the number of telomerasepositive cells should reflect the fraction of cycling cells, rather than the numt~r of cells actually synthesizing DNA at any one time. The sensitive telomere-repeat-amplification protocol can detect telomerase activity even if only 1:1000 to 1:10000 cells is telomerase positive. Although possible, it is unlikely that the failure to detect telomerase activity in many premalignant lesions is due to a reduced growth rate compared with malignant tumors. The presence of telomerase activit3' in CD34+CD38 + stem cells, and even in activated T and B cells, increases the likelihood that many hematological malignancies can, in fact, arise from telomerase-competent cells. There are different types and stages of stem cells Cancer is a disease of dividing cells. Most differentiated cells are postmitotic or dividing very slowly, therefore, the growth advantage of cells blocked from fully differentiating (or undergoing apoptosis) would be expected. However, the pre~nce of poorly differentiated markers does not imply that the cancer arose from a telomerase-competent, primitive stem cell. Many late stem cells are telomerase negative [e.g. cultured human myoblasts m and basal epidermal cells from sun-protected skin (S. Taylor et al., unpublished)l, .so that the potential derivation of tumors from stem cells does not imply that they
DEBATE
arise without the need for telomemse reactivation. Models for the o r i g i n of
tclomerase-positive cancer cells With these qualifications in mind, one can consider the following sampling of many potential scenarios for the origin of telomemse-positive cancer cells. The likelihood of a mutation incr~ises with populatkm size. In most tissues, the number of tdomerase-negative stem cells is likely to exceed vastly the number of telomemse-positive, primitive stem cells and, thus, these cells are likely to be the target populatkm for the accumulation of mutations. It is generally helieved that a series of clonal expansions is required to accumulate the mnhiple abnormalities of the malignant cell. Teiomerase reactivation cotfld occur during any of these expansions. By analogy with what has been obse~'ed in cultured cells, we wot, ld predict that the first mutations would be ones that pennined ckmal expansion in the context of the normal 'toni:tel inhibited' environment of normal tissues. As telomems shorten, mutations in other genes (e.g. the genes encoding p53 and RB) accumulate t':, which result in genomic instability, an extended lifespan and continued, progressive erosion oflelt)lneres t~-t-.-'a. At this ix)int. end-to-end chromosome fusions are frequently obse~'ed concomitantly with critically shortened telomeres. These events could contribute to the loss of hctemzygosity and the expression of recessive mutations. w h i d l result in the rcactivaticm of telomerase and stabilizatkm of telomere length. ;is well as fixation of the additional mut:ttkms required for invasiveness and metastasis. The lack of detectable telomerase activity in many benign and premalignant lesitmss (:is well as in cell culture models I~elore immortalizationlS-n-). tile presence of short telomcres in many tulnors 12-1t and tile presence of telomerase-negative primary tunlors 21.22 {some of w h i d l exhihit telomer;ise-positive nletastases22) ale all consistent wRh this scenario. In addition, cell hybrids made by fusing tdomcrase-expmssing human cells with normal diplffid human cells (with no detectable telomemse activity) result in tl-t~ i,lhil)itkm uf telomerase activity and the eventual cessation of proliferation nn. Reintroduction
of a normal human chromosome into a tumor cell line that is expressing telomerase also results in the inhibition of telomerase activity, progressive telomere shortening and the eventual cessation of cell proliferation 23. These results are consistent with the presence of gene products that regulate the activity oftelomerase and support the notion that loss or mutation of such regulatory genes could result in the reactivation of telomerase activity during cancer progression. An alternate scenario is that a telomerase-positive stem cell represents the progenitor cell :~." cancer. Compensating for tile lack .: a large population, in which mutations could occur, would be the potential elimination of several mutagenic steps needed to overcome o~l!ular. =nescence and tile reactivation of r .. "merase The fact that some vep, early premalignant lesions are telomemse positive"~27 is consistent with this pathway. In order for a premalignant lesion derived from a telomerasepositive stem cell to be telomerase silent, one would have to postulate that tile overwhelming majority of the cells in the iesion represented moredifferentiated telomerase-negative progeny of the actual cell leading to cancer; and that the Ix)pulation size of these precursor ceils of cancer was, nonetheless, large enough to permit sufficient secondary and tertiatT mutations to occur to pemlit cancer progression.
Telomera._~ activity d u r i n g development Telomerase activity is present in many tissues throughout chicken (R.I). Rantcriz et ai.. unpublished) and human l~tal development is. and, thus, the pool size for tile development of additional mutations is large at that time. However, this activity Iu2conles undetectable in nit)st tissues before or shortly after birth. Because telomemse activity is wi'despread during early development, we suspect that many childhood malignancies might rest, It from a failure to repress telolnerase, whereas most adult tuntors would result from a reactivatkm of telomemse activity.
Conclusions In stunmary, We helieve cancer is a diverse set of diseases and that the scenario discussed by Greaves in the TIG AeRtt. 1996 VOL. 12 No. 4
130
preo:ding article is likely to occur in som,:' situations. The complexities of the expression, and regulation, of telomerase and telomere length do not permit a rigorous distinction between the two pathways at this time. Nonetheless, w e believe that much of the evidence favors the interpretation that most adult tumors develop from telomerase-negative precursors,
Acknowledgements Work in the authors' laixwatoD" is supported by research grants from the NIH (AG079-)2).Geron Cx)rporation,Susan Komen Breast Cancer Foundation. Cap Cure and the Te.'os Advanced Technolog3" Program (003660-041 ).
References I Harley, C.B., Futcher, B.A. and Greider, CAV. (1990) Xature3i5, 458-460 2 Greider, C.W. (1990) Bioessto's 12, 363-369 3 Harley. C.B. ( 1991 ) Murat. Res. 2"~6, 271-282 4 Harley, C.B. (1995)./. ,~?allnst. Health Rt~'. 7, 64-68 5 AIIsopp. R.C. et al. (1992) Proc. A2ttl Acad. Sci. USA89,1011,i-10118 6 Wright, W.E. and Shay, J.W. (1995) Trends Cell BioL 5, 293-297 7 Shay, J.W., Werbin. tL and Wright, W.E. (1995) Can.J. Aging 14, 511--324 8 Kim, N.W. et al. (199~) Science 266. 2011-2015 9 Shay. J.W. (19951Mol. Med. Tod¢O, 1.378-384 10 Shay.J.W. and Wright, W.E. (1996) Cltrr. Opill. Oncol. 8. 66-71 11 Wright, W.E., llrasiskyte, D.. Piatyszek. M.A. and Shay. J.W. FIIBOJ. tin press~ 12 deLange, T. et al. (1990) MoL CeIL Biol. 10, 518-527 13 Hastie. N.D. et aL (1990) Nature 346. 866-868 J4 Hiyama, E. el aL (1995) hlt..L Oncol, 6,13-16 15 Counter. C.M. et aL t 19921 FAIBOJ. I1, 1921-1929 16 Shay.J.W.. Wright. W.E.. llrasiskyt, D. and Van Der Haegen, B.A. (1993) OncogeneS, 1407-1413 17 Shay, J.W., Tumlinson, G., Piatyszek. M.A, and Gollahon, L.S. (19951 MoL Cell. Biol. 15. 425-432 18 Wright. W.E et al. Dee. Get. (in press) 19 Shay,J.W., Pcreira-Smith, O.M. and Wright, W.E. (1991)/'-'~p. Cell Res. 196, 35-39 2 0 Klingelhutz, AJ. et aL (1994) MoL Cell BioL 14,961-969
DEBATE 21 Hiyama, E. et al. (1995) Nat. Med. 1, 249-257 22 Hiyama, K. et al. (1%95)J. Nat/ Cancer htst. 87, 895-902 23 Ohmum, H. a a/. (1995)Jpn.J.
Cancer Res. Rapid Cont. 86, 899-904 24 Taham, H. et al. (1995) C/in. Cancer Res. RapM C~m. 1,1245-1251 25 Hiyama, E. et al. (1~)5) CancerRes.
TECHNICAL
455, 3258-3262 26 Hiyama, E. et aL (I996)J. Nat/ Cancer hist. 8g, 116-122 27 Tahara, H. el al. (1995) Cancer Res. 55, 2734-2736
TIP
R a p i d m a p p i n g o f m R o c h o n d r i a l DNA deletions b y large-h--agmeat ]PI2B.
I
Mapping mitochondrial DNA (mtDNA) deletions in patients with 1 2 3 4 mitochondrial myopathies requires Southern blot analysis with different restriction enzymes and different mtDNA probes. These delimited breakpoint regions are then amplified as a short PCR 21 kb >,product forfurtherchamcterizationbysequenceanalysis.T~efirst 12 kb ~ ~ 15.5 kb step is time-consuming and needs several micrograms of total 13.5 kb DNA, which are difficult to obtain from muscle biopsies that are [ small. Recent reports described amplification of large PCR frag- ] ments up to 20kb (Refs 1, 2). We report a simple procedure to i amplify normal and deleted mtDNA that allows a rapid characterization of the rearrangements in patients. Isolation of total DNA from frozen muscle was performed under standard conditions3. PCR reactions were carded out using a set of primers within the mtDNA noncoding region and part of the phenylalanine tP~NIA coding sequence: XLI 5' 5 kb >.CCCACAG'ITrATGTAGCITACCTCCI'CA3' (nucleotides 571-598) and XL2 5' TrGATI'GCTGTACTFGCTI'GTAAGCATG 3' (nucleotides 16220-16193). We used the Expand Long PCR System (Boehringer FlcamE 1. The genome of the human mitochondrial DNA Mannheim). Two separate master mixes were prepared in 25 ~l: was amplified from a control (lane 3) and a patient (lane 4). one with 0.5 ngl of each dNTP and 0.3 I~Mof each primer, and the The molecular weight markers are 1 kb ladder (GibcoBRL) other with 250 ng of template DNA, 5 Id of 10X PCR buffer 3 (lane 1) and HindII!-EcoRI restricted h DNA (lane 2). (22.5 trot MgCI2 and detergents) and 0.75 ~l of the enzyme mix containingthermost2ble Taq and Pwo DNA polymerases. The two mixes were added to a microamplificationtube with a final volume of 501xl.Amplification reactions were performed in a GenAmp PCR System 480 (Perkin-Elmer) as follows: one cycle of 4rain at 93°C, 30 cycles of 10s az 93°C, 30s at 62°C and 13min at 68°C (during the last ten cycles, 20 s per cycle were added to the extension step) followed by a 10 min hold at 72°C. PCR products (2 fl,l) were analysed on 0.6-0.8% standard agarose gels (Biomd), which were mn overnight. We obtained clean 15,5 kb products from controls. A second shorter fragment (approximately 13.5 kb) was amplified from a patient beating a deleted mtDNA population (Fig. 1) showing heteroplasmia. All PCR products were separately digested by &;tc'Iand ECoRV(Fig. 2a). The fragments expected (9077 and 6572 bp with Sad, and 6137, 3555, 3346 and 2611 bp with EcoRV)were observed in the controls and the patient (Fig. 2b). (a)
1
2
3
4
5
6
7
8
(b)
I i i [ F~----q- ~ [ -F--I-- 111 i ~ ~ 1 2 3 4 5 6 7 8 9 10 12 13 14 15 16
kb
21 kb >.1 2 k b >.-
bp
9077
6572
Sa~l(. . . . Wild-lype 5kb
I
)
I[ ~ =
>-
4kb
>.-
3kb
>.-
EcoFIV(hi a192)
lop
2611
3555
Ec~V (hi 6737)
6137
EcaRV(nt 128'4)
3345
Ft(;trm.:2. (a) Digestion of PCR products by Sad and EcoRV. Lane 1 shows the molecular weight markets are 1 kb ladder and lane 2 shows the Hi~dlll-Eco_RI mstrict..ed h DNA. lane 3 shows Lhe Sad digestion of PCR product2: from file patient and lanes 4 and 5 show the Sad digestion of PCR products from the controls. Lane 6 shows the EcoRVdigestion from the patient and lanes 7 and 8 show the EcoRVdigestion from the controls. (b) Restriction map of the wild-wpe PCR products. Abbreviation: tat, nucleotide. TIG APRIl, 1996 VOL. 12 NO. 4 C*~pyrlght ~e~l ,)~36El~,v k'r Science I~d. All righls le~,r~ ell. 01 (~4-9"32~ t)~i$ ISt~) PII: S{)If~C-952~(~XDi(RH)7-~
131
qke reactivation of telomerase activity in cancer progression JERRY W. SHAYAND WOODRING E. WRIGHT [email protected] [email protected] THEUNIVEtL~;ITYOFTEX.~.SSOVI'~'F.SlXRXMEDIC.~CENTF.LDEPART?,IEN'OF f CELLBIOLOGYA~XtNEUROSCIENCE,5323 HAaR'¢HINESBOULEVARD, DaLt~S,TX 75235-9039, USA.
There is increasing evidence suptx~rting the hypothesis that the shortening of telomeres is the clock that times cellular senescencet-7~ Most human primary tumors contain telomerase (the enzyme activity that prevents telomere shortening), while most normal tissues and cells lack this activity (Ref. 8 and reviewed in Refs 9,10). These observations contribute to the concept that normal cells lack sufficient proliferative capacity to undergo the repetitive cycles of mutation and clonal expansion that would be required to accumulate the multiple genetic abnormalities present in cancer cells. In most cases, escape from the proliferative limitations of cellular senescence by telomerase reactivation might be a prerequisite for the development of a malignant tumor cell. Although we believe that this scenario has a great deal of experimental support and is likely to represent real biology, there are a variety of subtleties that need to be considered. One of these, raised by Mel Greaves in the preceding article, which we will consider below, is that telomerase-positive cancer cells arise from tdomerase-positive stem cells with out the need for a reactivation step.
How does telomerase regulate telomere length? Telomere length is likely to be a good indicator of the proliferative history of telomerase-negative cells, in which telomeres shorten progressively with each :,ucceeding cell division 1-'~. However, telomere length might not be informative in telomerase-pcsitive cells. The 'appropriate' level of telomerase activity in any cancer cell should maintain a stable telomere length, regardless of whether the telomeres are 2 or 20kb long. Increased or decreased functional telomevase activity should result in a changing telomere length. At present, virtuaily nothing is known about the mechanism by which cells expressing telomerase regulate the length of their
telomeres n. The presence of shoa telomeres in many tumors 12-14 suggests that considerable telomere shortening occurred before the telomerase was reactivated, but clearly does not prove it, because telomere shortening could have continued in the presence of telomerase. By the same token, tumors with long telomeres need not be derived from stem cells that never repressed telomerase and, thus, never shortened their telomeres, because the telomeres could have been elongated after telomerase reactiw~tion. In both cases, the result miglt reflect a regulated process rather than a dysfunctional enzyme, because an appropriate ~ctivity must be obtained if a stabh~ telomere length is ultimately to be n:aintained. Telomere length in telomerase-positive tumor cells is, thus. not an effective discriminator about whether or not the cancer progenitor cell was a telomerase-positive stem cell. Regulation of telomerase activity The evaluation of telomerase is further complicated by the fact that telomerase-positive cells become telomerase-silem when they become quiescent (S. Holt et al., unpublisbed). This is not surprising, because an extraordinarily tight regulation of intracellular activity would be required to prevent telomere elongation in a telomerase-competent stem cell or germ cell that remained quiescent for many years. This observation has a variety of implications. On the one hand, it suggests that the most primitive stem cells that rarely divide might be protected from the effects of any anti-telomerase cancer therapies. On the other hand, it implies that evidence for the lack of telomerase achvi W in normal or tumor cells needs to be carefully interpreted. Normal diploid cells dividing in culture 15--E7 and hyperproliferative benign lesions, such as leiomyomass, do no: exhibit TIG APRIL 1996 VOL. 12 NO. 4
C~)pjxight©19%Et~'vlcrScicno:lld. Allnght.,reservcd. Ol(~9525/96/$15.00
129
telome~ose activity, so tim lack of telomerase in these situations clearly does not reflect the lack of mitotic activity. However, it is possible that the lack of detectable telomerase activity in normal tissues and some benign or premalignant lesions could reflect the lack of cell division, rather than the lack of telomerase-competent cells. Because telomerase activi W is detected in all phases of the cell cycle in immortalized cells and is only absent in GO (S. Holt et al., unpublished), the number of telomerasepositive cells should reflect the fraction of cycling cells, rather than the numt~r of cells actually synthesizing DNA at any one time. The sensitive telomere-repeat-amplification protocol can detect telomerase activity even if only 1:1000 to 1:10000 cells is telomerase positive. Although possible, it is unlikely that the failure to detect telomerase activity in many premalignant lesions is due to a reduced growth rate compared with malignant tumors. The presence of telomerase activit3' in CD34+CD38 + stem cells, and even in activated T and B cells, increases the likelihood that many hematological malignancies can, in fact, arise from telomerase-competent cells. There are different types and stages of stem cells Cancer is a disease of dividing cells. Most differentiated cells are postmitotic or dividing very slowly, therefore, the growth advantage of cells blocked from fully differentiating (or undergoing apoptosis) would be expected. However, the pre~nce of poorly differentiated markers does not imply that the cancer arose from a telomerase-competent, primitive stem cell. Many late stem cells are telomerase negative [e.g. cultured human myoblasts m and basal epidermal cells from sun-protected skin (S. Taylor et al., unpublished)l, .so that the potential derivation of tumors from stem cells does not imply that they
DEBATE
arise without the need for telomemse reactivation. Models for the o r i g i n of
tclomerase-positive cancer cells With these qualifications in mind, one can consider the following sampling of many potential scenarios for the origin of telomemse-positive cancer cells. The likelihood of a mutation incr~ises with populatkm size. In most tissues, the number of tdomerase-negative stem cells is likely to exceed vastly the number of telomemse-positive, primitive stem cells and, thus, these cells are likely to be the target populatkm for the accumulation of mutations. It is generally helieved that a series of clonal expansions is required to accumulate the mnhiple abnormalities of the malignant cell. Teiomerase reactivation cotfld occur during any of these expansions. By analogy with what has been obse~'ed in cultured cells, we wot, ld predict that the first mutations would be ones that pennined ckmal expansion in the context of the normal 'toni:tel inhibited' environment of normal tissues. As telomems shorten, mutations in other genes (e.g. the genes encoding p53 and RB) accumulate t':, which result in genomic instability, an extended lifespan and continued, progressive erosion oflelt)lneres t~-t-.-'a. At this ix)int. end-to-end chromosome fusions are frequently obse~'ed concomitantly with critically shortened telomeres. These events could contribute to the loss of hctemzygosity and the expression of recessive mutations. w h i d l result in the rcactivaticm of telomerase and stabilizatkm of telomere length. ;is well as fixation of the additional mut:ttkms required for invasiveness and metastasis. The lack of detectable telomerase activity in many benign and premalignant lesitmss (:is well as in cell culture models I~elore immortalizationlS-n-). tile presence of short telomcres in many tulnors 12-1t and tile presence of telomerase-negative primary tunlors 21.22 {some of w h i d l exhihit telomer;ise-positive nletastases22) ale all consistent wRh this scenario. In addition, cell hybrids made by fusing tdomcrase-expmssing human cells with normal diplffid human cells (with no detectable telomemse activity) result in tl-t~ i,lhil)itkm uf telomerase activity and the eventual cessation of proliferation nn. Reintroduction
of a normal human chromosome into a tumor cell line that is expressing telomerase also results in the inhibition of telomerase activity, progressive telomere shortening and the eventual cessation of cell proliferation 23. These results are consistent with the presence of gene products that regulate the activity oftelomerase and support the notion that loss or mutation of such regulatory genes could result in the reactivation of telomerase activity during cancer progression. An alternate scenario is that a telomerase-positive stem cell represents the progenitor cell :~." cancer. Compensating for tile lack .: a large population, in which mutations could occur, would be the potential elimination of several mutagenic steps needed to overcome o~l!ular. =nescence and tile reactivation of r .. "merase The fact that some vep, early premalignant lesions are telomemse positive"~27 is consistent with this pathway. In order for a premalignant lesion derived from a telomerasepositive stem cell to be telomerase silent, one would have to postulate that tile overwhelming majority of the cells in the iesion represented moredifferentiated telomerase-negative progeny of the actual cell leading to cancer; and that the Ix)pulation size of these precursor ceils of cancer was, nonetheless, large enough to permit sufficient secondary and tertiatT mutations to occur to pemlit cancer progression.
Telomera._~ activity d u r i n g development Telomerase activity is present in many tissues throughout chicken (R.I). Rantcriz et ai.. unpublished) and human l~tal development is. and, thus, the pool size for tile development of additional mutations is large at that time. However, this activity Iu2conles undetectable in nit)st tissues before or shortly after birth. Because telomemse activity is wi'despread during early development, we suspect that many childhood malignancies might rest, It from a failure to repress telolnerase, whereas most adult tuntors would result from a reactivatkm of telomemse activity.
Conclusions In stunmary, We helieve cancer is a diverse set of diseases and that the scenario discussed by Greaves in the TIG AeRtt. 1996 VOL. 12 No. 4
130
preo:ding article is likely to occur in som,:' situations. The complexities of the expression, and regulation, of telomerase and telomere length do not permit a rigorous distinction between the two pathways at this time. Nonetheless, w e believe that much of the evidence favors the interpretation that most adult tumors develop from telomerase-negative precursors,
Acknowledgements Work in the authors' laixwatoD" is supported by research grants from the NIH (AG079-)2).Geron Cx)rporation,Susan Komen Breast Cancer Foundation. Cap Cure and the Te.'os Advanced Technolog3" Program (003660-041 ).
References I Harley, C.B., Futcher, B.A. and Greider, CAV. (1990) Xature3i5, 458-460 2 Greider, C.W. (1990) Bioessto's 12, 363-369 3 Harley. C.B. ( 1991 ) Murat. Res. 2"~6, 271-282 4 Harley, C.B. (1995)./. ,~?allnst. Health Rt~'. 7, 64-68 5 AIIsopp. R.C. et al. (1992) Proc. A2ttl Acad. Sci. USA89,1011,i-10118 6 Wright, W.E. and Shay, J.W. (1995) Trends Cell BioL 5, 293-297 7 Shay, J.W., Werbin. tL and Wright, W.E. (1995) Can.J. Aging 14, 511--324 8 Kim, N.W. et al. (199~) Science 266. 2011-2015 9 Shay. J.W. (19951Mol. Med. Tod¢O, 1.378-384 10 Shay.J.W. and Wright, W.E. (1996) Cltrr. Opill. Oncol. 8. 66-71 11 Wright, W.E., llrasiskyte, D.. Piatyszek. M.A. and Shay. J.W. FIIBOJ. tin press~ 12 deLange, T. et al. (1990) MoL CeIL Biol. 10, 518-527 13 Hastie. N.D. et aL (1990) Nature 346. 866-868 J4 Hiyama, E. el aL (1995) hlt..L Oncol, 6,13-16 15 Counter. C.M. et aL t 19921 FAIBOJ. I1, 1921-1929 16 Shay.J.W.. Wright. W.E.. llrasiskyt, D. and Van Der Haegen, B.A. (1993) OncogeneS, 1407-1413 17 Shay, J.W., Tumlinson, G., Piatyszek. M.A, and Gollahon, L.S. (19951 MoL Cell. Biol. 15. 425-432 18 Wright. W.E et al. Dee. Get. (in press) 19 Shay,J.W., Pcreira-Smith, O.M. and Wright, W.E. (1991)/'-'~p. Cell Res. 196, 35-39 2 0 Klingelhutz, AJ. et aL (1994) MoL Cell BioL 14,961-969
DEBATE 21 Hiyama, E. et al. (1995) Nat. Med. 1, 249-257 22 Hiyama, K. et al. (1%95)J. Nat/ Cancer htst. 87, 895-902 23 Ohmum, H. a a/. (1995)Jpn.J.
Cancer Res. Rapid Cont. 86, 899-904 24 Taham, H. et al. (1995) C/in. Cancer Res. RapM C~m. 1,1245-1251 25 Hiyama, E. et al. (1~)5) CancerRes.
TECHNICAL
455, 3258-3262 26 Hiyama, E. et aL (I996)J. Nat/ Cancer hist. 8g, 116-122 27 Tahara, H. el al. (1995) Cancer Res. 55, 2734-2736
TIP
R a p i d m a p p i n g o f m R o c h o n d r i a l DNA deletions b y large-h--agmeat ]PI2B.
I
Mapping mitochondrial DNA (mtDNA) deletions in patients with 1 2 3 4 mitochondrial myopathies requires Southern blot analysis with different restriction enzymes and different mtDNA probes. These delimited breakpoint regions are then amplified as a short PCR 21 kb >,product forfurtherchamcterizationbysequenceanalysis.T~efirst 12 kb ~ ~ 15.5 kb step is time-consuming and needs several micrograms of total 13.5 kb DNA, which are difficult to obtain from muscle biopsies that are [ small. Recent reports described amplification of large PCR frag- ] ments up to 20kb (Refs 1, 2). We report a simple procedure to i amplify normal and deleted mtDNA that allows a rapid characterization of the rearrangements in patients. Isolation of total DNA from frozen muscle was performed under standard conditions3. PCR reactions were carded out using a set of primers within the mtDNA noncoding region and part of the phenylalanine tP~NIA coding sequence: XLI 5' 5 kb >.CCCACAG'ITrATGTAGCITACCTCCI'CA3' (nucleotides 571-598) and XL2 5' TrGATI'GCTGTACTFGCTI'GTAAGCATG 3' (nucleotides 16220-16193). We used the Expand Long PCR System (Boehringer FlcamE 1. The genome of the human mitochondrial DNA Mannheim). Two separate master mixes were prepared in 25 ~l: was amplified from a control (lane 3) and a patient (lane 4). one with 0.5 ngl of each dNTP and 0.3 I~Mof each primer, and the The molecular weight markers are 1 kb ladder (GibcoBRL) other with 250 ng of template DNA, 5 Id of 10X PCR buffer 3 (lane 1) and HindII!-EcoRI restricted h DNA (lane 2). (22.5 trot MgCI2 and detergents) and 0.75 ~l of the enzyme mix containingthermost2ble Taq and Pwo DNA polymerases. The two mixes were added to a microamplificationtube with a final volume of 501xl.Amplification reactions were performed in a GenAmp PCR System 480 (Perkin-Elmer) as follows: one cycle of 4rain at 93°C, 30 cycles of 10s az 93°C, 30s at 62°C and 13min at 68°C (during the last ten cycles, 20 s per cycle were added to the extension step) followed by a 10 min hold at 72°C. PCR products (2 fl,l) were analysed on 0.6-0.8% standard agarose gels (Biomd), which were mn overnight. We obtained clean 15,5 kb products from controls. A second shorter fragment (approximately 13.5 kb) was amplified from a patient beating a deleted mtDNA population (Fig. 1) showing heteroplasmia. All PCR products were separately digested by &;tc'Iand ECoRV(Fig. 2a). The fragments expected (9077 and 6572 bp with Sad, and 6137, 3555, 3346 and 2611 bp with EcoRV)were observed in the controls and the patient (Fig. 2b). (a)
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(b)
I i i [ F~----q- ~ [ -F--I-- 111 i ~ ~ 1 2 3 4 5 6 7 8 9 10 12 13 14 15 16
kb
21 kb >.1 2 k b >.-
bp
9077
6572
Sa~l(. . . . Wild-lype 5kb
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)
I[ ~ =
>-
4kb
>.-
3kb
>.-
EcoFIV(hi a192)
lop
2611
3555
Ec~V (hi 6737)
6137
EcaRV(nt 128'4)
3345
Ft(;trm.:2. (a) Digestion of PCR products by Sad and EcoRV. Lane 1 shows the molecular weight markets are 1 kb ladder and lane 2 shows the Hi~dlll-Eco_RI mstrict..ed h DNA. lane 3 shows Lhe Sad digestion of PCR product2: from file patient and lanes 4 and 5 show the Sad digestion of PCR products from the controls. Lane 6 shows the EcoRVdigestion from the patient and lanes 7 and 8 show the EcoRVdigestion from the controls. (b) Restriction map of the wild-wpe PCR products. Abbreviation: tat, nucleotide. TIG APRIl, 1996 VOL. 12 NO. 4 C*~pyrlght ~e~l ,)~36El~,v k'r Science I~d. All righls le~,r~ ell. 01 (~4-9"32~ t)~i$ ISt~) PII: S{)If~C-952~(~XDi(RH)7-~
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