angiogenesis , the formation of new blood vessels , is essential for tumor growth and metastasis   and inhibition of angiogenesis is an important new approach for therapy of many cancers     [ 1 , 2 ] . a principal regulator of angiogenesis is vascular endothelial growth factor ( vegf ) .
the vegf family is comprised of at least five genes ( vegf a through e ) , of which the most potent activators of angiogenesis are vegf - a and vegf - b     .
analysis of the vegf - a ( termed vegf here and elsewhere in the text ) gene promoter region has revealed numerous potential transcriptional activator sites     .
one of the best studied stimuli for vegf synthesis and secretion is hypoxia , which acts by upregulating hypoxia inducible factor-1 ( hif-1 ) .
inhibition of hif-1 may therefore decrease angiogenesis by reducing vegf levels and potentially other proangiogenic factors , such as angiopoeitin-1 and -2 ( ang-1 and ang-2 )     [ 68 ] , basic fibroblast growth factor ( bfgf ) , and platelet derived growth factor ( pdgf ) , all well known regulators of angiogenesis .
t - oligos , telomere homolog oligonucleotides , activate p53 and e2f1 , resulting in apoptosis .  
however , in p53 null cells like malignant melanoma an cells ( mm - an ) , apoptosis is induced by the p53 homolog p73 , presumably acting coordinately with e2f1 .
moreover , e2f1 has also been reported to inhibit angiogenesis     [ 9 , 12 ] . while e2f1 decreases vegf production in fibroblasts through p53 activation     , its effect on angiogenesis in cells that lack functional p53 , such as mm - an cells has not been studied     [ 11 , 13 ] .
several lines of evidence suggest that normal cells have an integrated program of genome - protective responses , functionally analogous to the bacterial sos response , that is based in the telomeres and appears to be abrogated in malignancy .
telomeres , the ends of chromosomes , are maintained in a loop configuration by insertion of the single - stranded 3 overhang into the proximal telomere duplex .
disruption of this loop structure by removal of the principal binding protein trf2 ( telomere repeat binding factor 2 ) leads to exposure and digestion of the overhang and activation of atm ( ataxia telangiectasia mutated ) and its effector protein p53 , followed by apoptosis or senescence , depending on cell type .
knockdown of another telomere - associated protein , the protection of telomeres-1 ( pot-1 ) , also expected to expose the ttaggg telomere repeat sequence , activates atr ( ataxia telangiectasia and rad3-related ) , leading to similar downstream effects     [ 16 , 17 ] .
moreover , treatment of malignant cells with rnai to knockdown the expression of ter , the rna subunit of telomerase , rapidly alters the expression of many genes in a pattern predicted to reduce cancer cell proliferation and invasiveness , then leads to apoptosis in a time course far too rapid to be attributable to the expected loss of telomerase activity and consequent telomere shortening , suggesting that other telomere - based effects are responsible .
our laboratory has described several anticancer properties of oligonucleotides homologous to the telomere repeat sequence ttaggg ( t - oligos )     [ 10 , 11 , 13 , 15 , 1928 ] .
t - oligos provided to cultured cells rapidly accumulate in the nucleus and mediate dna damage responses without digestion of the telomere overhang or other detectable effects on genomic dna     [ 10 , 13 , 15 , 19 , 23 , 25 , 26 , 29 ] .
t - oligos activate the atm kinase     [ 21 , 27 ] , upregulate and activate p53     [ 30 , 31 ] , as well as upregulate and/or activate its homolog p73 , e2f1 , p16 , p33 , p27 , and p95/nbs1 , and phosphorylate the histone variant protein h2ax     [ 11 , 13 , 21 , 22 , 24 , 25 ] .
in addition , t - oligos promote differentiation of melanoma cells and downregulate the inhibitor of apoptosis protein iap / livin in these cells .
t - oligo effects require wrn     , the protein mutated in the progeroid cancer - prone werner syndrome , and are associated with formation of classic dna damage foci at telomeres     . in combination , these signaling cascades result in induction of apoptosis , autophagy , and/or senescence selectively in cancer cells     [ 13 , 19 , 27 , 28 ] ; while in normal cells they lead to transient cell cycle arrest , increased dna repair capacity and adaptive differentiation     [ 19 , 21 , 23 , 2527 , 30 ] .  
complementary , unrelated , or scrambled oligonucleotides comparably accumulate in the nucleus , but do not cause dna damage - like signaling or affect growth , differentiation or survival of malignant cells     [ 11 , 13 , 15 , 19 , 21 , 24 , 27 , 28 ] . because the t - oligo - induced transcription factors p53 , p73 , and e2f1 are known to affect endothelial cell ( ec ) survival , differentiation , and proliferation during tumor angiogenesis    
[ 12 , 33 , 34 ] and because blocking angiogenesis would be an additional anticancer mechanism of action for t - oligos , we asked whether t - oligo treatment inhibits tumor angiogenesis .
we now report that t - oligo inhibits angiogenesis in the aggressive human melanoma cell line mm - an , derived from a metastatic melanoma     , by decreasing production and secretion of proangiogenic factors in both tumor cells and ecs . as well
, t - oligo treatment decreases the number of total and functional ( perfused ) vessels in flank tumors of mm - an cells in scid mice after two systemic injections .
human microvascular endothelial cells ( hmvecs ) and human umbilical vein endothelial cells ( huvecs ) were obtained at passage 2 and used by passage 46 .
cells were maintained in egm-2 medium with 2% fbs plus growth factors ( bullet kit ) ( cambrex bio sciences , walkersville , md ) . human melanoma mm - an and ep cells      were cultured in modified eagle 's medium ( mem ) ( mediatech , inc . ,
herndon , va ) supplemented with fetal bovine serum ( fbs 2% ) , calf serum ( cs 8% ) , and antibiotic - antimicotic .
human breast adenocarcinoma ( mcf-7 ) and ovarian adenocarcinoma ( ovcar3 ) cells were cultured and maintained according to atcc recommendations .
the oligonucleotide was synthesized by midland certified reagent ( midland , tx ) and then diluted from a 2  mm stock in medium to obtain a final concentration of 40  m .
mm - an , ep , mcf-7 , and ovcar3 cells were grown in their corresponding media and treated once with diluent or 40  m t - oligo . for qrt - pcr
the sequences used were as follows : e2f1 forward : cggtgtcgtcgacctgaact , e2f1 reverse : aggacgttggtgatgtcatagatg , e2f1 probe : tgccgaggtgctgaaggtgcag ; ang-1 forward : cagaaaacagtgggagaagatataacc , ang-1 reverse : tgccatcgtgttctggaaga , ang-1 probe : caacatgggcaatgtgcctacactttc ; ang-2 forward : ggctgggcaatgagtttgtc , ang-2 reverse : cccagtccttcagctggatct ; ang-2 probe : accggtcagcaccgctacgtgc .
cells were treated with diluent or 40  m t - oligo and harvested 0 , 1 , 16 , and 32 hours after treatment .
emsas were carried out as described   using a total of 5  g of nuclear protein per lane .
the assay was performed using consensus sequences of e2f1 and hif-1 transcription factors ( santa cruz biotechnology , ca ) .
the specificity of the bands was confirmed by using either 25 or 50 excess of cold probe as competitor and mutant oligos as control . as an additional negative control
, nuclear extracts were incubated with a specific competing e2f1 antibody before adding the radioactive ( p - labelled ) consensus oligonucleotides .
cells were treated with t - oligos , harvested at various times , snap - frozen , and stored at 70c .
total cellular protein was isolated and 50  g of total protein was processed for western blot analysis as described .
antibody reactions were performed with the following antibodies : anti - e2f1 ( neomarkers , inc . ,
fremont , ca ) , anti - vegf - a ( sc-507 , santa cruz biotechnology , inc , santa cruz , ca ) that recognizes all vegf - a isoforms , and anti - vegf - r2 ( a kind gift from dr .
nader rahimi , departments of ophthalmology and biochemistry , boston university school of medicine ) . the human vegf immunoassay kit ( quantikine , r&d systems , minneapolis , mn ) was used to compare the release of vegf into the medium by ecs and mm - an cells
each cell type was cultured in its appropriate medium and treated with either diluent or t - oligo ( 40  m ) .
the conditioned medium was collected at 24 , 48 , and 72 hours after treatment and frozen at 70c until it processed for elisa .
the plate was read using the tecan spectra model 96 well microplate reader ( mtx lab systems , vienna , va ) .
200  l of matrigel basement membrane matrix ( phenol - red free ) ( bd biosciences , bedford , ma ) was added per 4-well chamber slide and allowed to solidify for 1 hour at 37c .
huvecs ( 50,000 cells ) were then added to each chamber in 500  l of medium as described     .
cells were treated at the time of plating with diluent , 40  m , 80  m , or 120  m of t - oligo . some of the cells treated with 40  m of t - oligo were retreated a second time at 2 hours postplating , at the time tube - formation
some cells that were treated twice with 40  m of t - oligo received a third treatment 6 hours after plating .
tube - formation was observed and photographed at numerous time points starting from 2 hours and up to 24 hours after treatment . of note , after tubular structures formed in control wells , they stayed intact up to 72 hours ( data not shown ) .
image - tool ( the university of texas health science center in san antonio , san antonio , tx ) software was used to quantify the length of tubular structures . the biocoat matrigel invasion chamber ( bd biosciences , bedford ,
mm - an cells were treated with diluent or 40  m t - oligo and media were collected after 72 hours , based on elisa data ( mm - an conditioned media ) . conditioned mm - an medium was then used as the chemoattractant for the assay .
ecs and mm - an cells were grown on the inserts ( upper chambers ) and allowed to invade through the matrigel and attach to the membrane as described in the manufacturer 's protocol .
invasion was assessed after 22 hours by staining with diff - quick ( fisher , atlanta , ga ) bisected membranes from the bottom of the chambers ( containing the invading cells ) as described in the manufacturer 's protocol .   of note , we believe that any effect on ecs or mm - an invasion can not be attributed to residual active t - oligos in the conditioned medium , diffusing into the upper chamber and killing mm - an cells , because t - oligos are rapidly degraded in serum - containing medium . with a measured half - life for a 12-base telomere homolog of 46 hours
, the 16-base t - oligo would thus have been present in the medium after 72 hours of conditioning at an estimated ( 1/2 ) or 10-fold less than the initial therapeutic concentration .
2  10  mm - an cells were injected subcutaneously into the flank of 6 week old scid mice ( fox - chase cancer center , philadelphia , pa ) .
mice ( 5 - 6 per treatment group ) were injected via tail vein with t - oligo or diluent alone when tumors were first palpable ( 2 - 3  mm diameter ) .
tumor sizes were recorded every 2 - 3 days throughout the experiment using electronic callipers and all animals were sacrificed 4 weeks after tumor inoculation .
the diagnosis of melanoma was confirmed histologically on sections of each nodule cut through the center of the clinical tumor .
2  10  mm - an cells were injected subcutaneously into the flank of 6 week old scid mice ( fox - chase cancer center , philadelphia , pa )
. then mice ( 5 - 6 per treatment group ) were injected via the tail vein with t - oligo or diluent alone ( 60  nmoles / injection , 15  mg / kg ) when tumors were first palpable ( 2 - 3  mm diameter ) .
microvascular density ( mvd ) in bisected tumors were assessed 24 hours after two systemic injections of t - oligos by using two ec - specific markers cd31 ( pecam-1 )      and bandeurea simplicifolia ( bs)-1 lectin conjugated to rhodamine ( vector laboratories , burlingame , ca ) . to measure functional mvd in the tumor tissue ,
30 minutes before sacrifice a set of mice ( 5 per treatment group ) were anesthetized and were perfused with 0.5  mg ( in 100  l of isotonic solution ) of rhodamine - conjugated bs-1 lectin as described     [ 40 , 41 ] .   to measure total mvd , 6  m cross - sections of bisected tumor tissue ( of the same mice perfused with bs-1 lectin )
were stained with cd31 primary antibody followed by fitc - labelled secondary antibody as described     .  
samples were photographed using a multicolour fluorescence microscope ( nikon , nikon instruments inc , melville , ny ) , and analyzed using a digital image analysis system ( nikon ) .
anova with post hoc analysis by scheffe and bonferroni - dunn and unpaired t - test were performed using statview ( sas institute , inc . ,
mm - an cells were treated once with t - oligo or diluent alone , provided in fresh medium , and processed for western blot analysis .
compared to diluent , t - oligo decreased vegf protein level at 24 hours ( p < .01 ) by ~41% and at 48 hours ( p = ns ) by ~32% ( figures 1(a ) and 1(b ) ) .
in addition , after 48 hours t - oligo treatment reduced ang-1 mrna ( p < .01 ) by ~60% ( figure 1(c ) ) and ang-2 mrna ( p < .04 ) by ~45% ( figure 1(d ) ) .
similar decreases in vegf ( supplemental figures 1(a)1(c ) and ang-1 ( supplemental figures 2(a)2(c ) ) mrna were seen in a second human melanoma line ep , as well as in other malignant cell types . paired dishes of mm - an and ecs were treated with 40  m t - oligo or diluent alone once .
medium was collected after 24 , 48 and 72 hours and vegf level in the medium was measured by elisa . in ecs ( hmvec and huvec ) , there was a ~50%75% variable and statistically insignificant decrease in vegf release into the medium over time in both diluent- and t - oligo - treated cells ( data not shown ) .  
in contrast , total vegf release from mm - an cells in diluent - treated dishes rose progressively over 72 hours to ~600%   ( of 24-hours vegf levels ) , while vegf release in t - oligo - treated dishes was decreased ( p < .0008 ) by ~30% over the 72-hours experiment after a single dosing at time 0 ( figure 1(e ) ) .  
thus , t - oligo not only decreased vegf protein expression in tumor cells but also the release of this potent angiogenic factor into the surrounding culture medium .
e2f1 mrna expression was increased at 24 hours ( p < .0001 ) by ~41% and at 48 hours ( p < .0001 ) by ~100% in t - oligo - treated mm - an cells compared to control ( figure 2(a ) ) .
similar increases in e2f1 mrna were seen in a second human melanoma line ep , as well as in other malignant cell types ( supplemental figures 3(a)3(c ) ) .
t - oligo treatment also increased e2f1 protein level at 24 hours to ~218% and at 48 hours to ~286% ( p < .02 ) of control values ( figures 2(b ) and 2(c ) ) .
in addition , there was a doubling in e2f1 dna binding activity 32 hours after treatment compared to diluent - treated cells ( figures 2(d ) and 2(e ) , compare lane 3 versus 4 ) .
mm - an cells were treated once with t - oligo or diluent alone and nuclear proteins were harvested up to 32 hours after treatment to evaluate dna binding activities of the angiogenic transcription factor hif-1. there was ~80%95% decrease in hif-1 dna binding activity at 1 and 16 hours after treatments ( figures 2(f ) and 2(g ) , lanes 3 versus 4 , and 5 versus 6 , respectively ) .
these data suggest that t - oligo - mediated inhibition in dna binding activity of hif-1 transcription factor contributes to decreased expression of the angiogenic factors , vegf and ang-1 , whose promoters contain binding sites for hif-1     .
we used mm - an medium collected 72 hours after addition of t - oligo or diluent alone ( mm - an conditioned media : t - oligo cm or diluent cm ) as the chemoattractant for an in vitro invasion assay     .
mm - an and hmvecs were both evaluated for invasion through matrigel to determine if t - oligo treatment reduces the chemoattractant properties of mm - an cells .   as recommended by the manufacturer of the matrigel invasion kit , after 22 hours of incubation the invading cells were fixed , stained and counted for each membrane .
mm - an cells plated above t - oligo cm had a ~96% decrease in invasion ( p < .03 ) through matrigel ( figures 3(a ) and 3(b ) ) .
hmvecs plated on inserts coated with matrigel and exposed to t - oligo cm had a ~40% reduction in matrigel invasion compared to controls , but this did not reach statistical significance ( p < .08 ) ( figures 3(c ) and 3(d ) ) .
these findings demonstrate that t - oligos reduce the migration / invasion of mm - an cells towards chemoattractant stimuli and , possibly , to a lesser degree affect hmvecs .
this is consistent with the observation that mm - an cells elaborate factors that promote migration and invasion of tumor cells , like vegf and ang-1 , and that these factor(s ) are reduced as a result of t - oligo treatment . because vegf signaling through vegfr-2 is principally responsible for ec survival , proliferation , migration , and angiogenesis     [ 4447 ] , we examined the effect of t - oligo treatment on these proteins .
hmvecs and huvecs were grown and treated with 40  m t - oligo or diluent alone .
compared to diluent - treated control cells there was a decrease ( p < .004 ) of ~86% in vegf expression at 24 hours and an insignificant decrease of ~50% at 48 hours in t - oligo - treated samples ( figures 4(a)4(c ) ) .
in addition , in t - oligo - treated hmvecs and huvecs there were ~29% ( p = ns ) and ~59% ( p < .04 ) reduction in the protein level of vegfr-2 at 24 and 48 hours , respectively ( figures 4(d)4(f ) ) .
thus , t - oligo - mediated antiangiogenic effects on ecs may be , in part , due to inhibition of vegf - vegr2 axis .
hmvecs were plated on matrigel in 4-well chamber slides in serum - containing medium and treated with diluent alone or increasing concentrations of t - oligo .
tube - like structure formation was maximal in controls at 22 hours and formal comparisons were made after 222 hours , as customary for this assay . by 22 hours , in cells treated once with either 40  m or 80  m t - oligo there was a ~19% reduction in average tube length compared to cells treated with diluent alone ( p = ns ) ( figures 5(a ) and 5(b ) ) .
cells treated with two separate doses of 40  m t - oligo at plating and 2 hours after plating showed a ~35% reduction in average tube length ( p < .03 ) .
cells treated once at plating with 120  m t - oligo showed a ~58% reduction in average tube length compared to the diluent - treated cells ( p < .001 ) . a reduction of ~83% ( p
< .0001 ) in tube length was observed in cells treated with 3 separate doses of 40  m t - oligo ( 40  m 3 ) added to the medium at the time of plating and then at 2 and 6 hours ( figures 5(a ) and 5(b ) ) .
these results indicate that t - oligo inhibits ec function in a manner dependent on the dose and frequency of administration , with the caveat that the very large amount of oligos administered , rather that specific telomere - based t - oligo initiated signalling may have contributed to the effect observed . in our future experiments
, we plan to examine the effect of large and/or fractioned t - oligo doses on survival of ecs . in melanoma tumors inoculated into scid mice , we determined the effect of systemic t - oligo injection on melanoma angiogenesis by evaluating functional and total vessel density in tumor tissue ( 5 mice per group ) . when tumors became palpable
( 2 - 3  mm in diameter , day 5 to 14 after inoculation ) we injected t - oligo ( 60  nmoles / injection , 15  mg / kg ) via the tail vein ( iv ) and injected again after 6 hours , then harvested tumors 24 hours after the second t - oligo injections .
representative images of tumor cross - sections were immunostained to identify all vessels as well as functional ( patent ) vessels 24 hours after t - oligo injection ( figure 6(a ) ) .   compared to control - injected mice
.002 ) in functional vessels in t - oligo - injected mice ( figure 6(b ) ) .  
there was also more than ~60% decrease ( p < .004 ) in total vessels in t - oligo - injected mice ( figure 6(c ) ) .  
these data corroborate our in vitro findings ( figures 4 and 5 ) and demonstrate that two systemic administrations of t - oligo reduce tumor angiogenesis and vessel patency in vivo ( figures 6(a)6(c ) ) .   to determine the effect of systemic t - oligo treatment on melanoma growth , in scid xenograft model mm - an cells
were injected subcutaneously in the flank . when tumors became palpable ( 2 - 3  mm in diameter , day 5 to 14 ) ,
the mice received daily systemic injections of t - oligo ( 60  nmoles / injection , 15  mg / kg bid ) or vehicle for 5 days only . in control animals , tumors increased in volume from 6.96  2.9  mm on day 6 to 88.04  19.72  mm on day 27 ( figure 6(d ) , blue line ) .
in contrast , in t - oligo treated animals , whose tumors were the same size as in controls on day 6 ( 6.48  2.57 ) there was very little tumor growth through day 15 ( 6.48  2.57 versus 9.92  3.8  mm ) and thereafter a slower and statistically insignificant increase in volume to 29.65  10.47  mm by day 27 ( figure 6(d ) , pink line ) .
thereby , in t - oligo - treated mice tumor growth was inhibited ( p < .003 ) by ~53% after ~4 weeks , when the experiment was terminated .
these data demonstrate that systemic administration of t - oligo for only 5 days has a persistent inhibitory effect on melanoma growth .
we have also evaluated t - oligos toxicity in several internal organs of scid mice 24 hours after the last iv injections ( 15  mg / kg bid for 5 days ) .
no systemic toxicity was observed in bone marrow , liver , intestines , brain , lungs and kidneys of t - oligo - injected mice ( figures 7(a)7(f ) ) .
tumor angiogenesis is essential for tumor growth and metastasis .   without active angiogenesis tumor diameter
angiogenesis is mediated through release of angiogenic factors by tumor cells and cells in the tumor stroma and microenvironment including , but not limited to , endothelial cells .  
we now report that telomere homolog oligonucleotides ( t - oligos ) decrease the synthesis and release of angiogenic factors by ecs and melanoma cells , inhibit ec tubulogenesis and impede melanoma cells and ecs from invading matrix ( matrigel ) .
ec proliferation , in vitro tubulogenesis , and survival are all known to be stimulated in large part by vegf . decreased
vegf levels or inhibition of receptor activation in ecs often correlate with decrease in tumor size and metastatic potential .
vegf binds to the extracellular domain of the vegfr-1 ( flt-1 ) and vegfr-2 ( flk-1 ) , inducing receptor dimerization and activation of tyrosine kinases by autophosphorylation , leading to angiogenesis , increased vascular permeability , and ec proliferation and survival .
we found that t - oligo decreases the expression of vegfr-2 by hmvec and huvec ( figures 4(c ) and 4(f ) ) .  
other investigators reported that receptor tyrosine kinase inhibitors ( tkis ) such as sunitinib and dasatinib reduce signaling through the raf / mek / erk pathway that is activated by ligand binding to angiogenic receptors like vegfr-2 , pdgfr- , fh-3 and c - kit , indirectly inhibiting tumor growth by affecting tumor angiogenesis .   by reducing vegf - r2 level ,
vegf also induces leakage within tumor vessels , allowing tumor cells to infiltrate blood vessels and migrate into the blood stream .
hence , changes in angiogenic factors even early in tumor formation can affect metastasis and spread   and inhibiting vegf production by t - oligos would be expected to reduce the metastatic potential of tumor cells .   additionally , increased blood vessel permeability within the tumor may interfere with adequate delivery and retention of chemotherapeutic agents     [ 54 , 55 ] .  
indeed , certain antiangiogenic agents that prevent tumor vessel leakage ( a phenomenon called  vessel normalization  ) were shown to enhance the delivery of chemotherapeutic agents into tumors     .  
thus , combination treatment with antiangiogenic factors together with conventional chemo - therapeutic agents may be superior to using the latter alone .  
furthermore , because vegf is likely required for migration and recruitment of ecs , t - oligo - me diated vegf reduction would also likely decrease the number of blood vessels in the tumor     [ 1 , 6 ] .  
our present findings suggest that t - oligos may induce potent antiangiogenic effects , enhancing their attractiveness as a therapeutic option for cancer .
in addition to vegf and its receptors , angiopoietin 1 and 2 ( ang-1 and ang-2 ) and their tyrosine kinase receptor tie-2 have been identified as major players in the processes of growth and remodelling of tumor vasculature     [ 57 , 58 ] .
although under certain conditions ang-2 may inhibit ang-1 effect , in an in vivo mouse model for melanoma      and in glioma cells      increased expression of ang-2 is thought to stimulate angiogenesis .   another angiogenic factor
, hif-1 , a transcription factor that is activated by hypoxia , exerts its effect by upregulating vegf levels .  
we found that t - oligo treatment inhibits hif-1 activity and ang-1 and ang-2 expression in melanoma cell line and decreases vegf synthesis secretion in these cells , strongly suggesting that t - oligo - mediated effects on tumor angiogenesis are transcriptional and ultimately affect several angiogenic molecules .
our laboratory has previously shown that t - oligo increases p73 level in the p53 null mm - an cells and that blocking p73 expression by rnai decreases t - oligo - induced apoptosis in these cells .   like p53 , p73 is known to inhibit angiogenesis , primarily through vegf down - regulation     .
therefore , we assume that in mm - an cells t - oligo decreases vegf production , in addition to its effect on hif-1 , by activating p73 through induction of e2f1 .  
however , in cells with functional p53 , we assume that t - oligo - induced p53 and p73 would cooperate to inhibit angiogenesis .  
furthermore , e2f1 is known to induce apoptosis in part by its effect on p73 .  
thus , induction of e2f1 by t - oligos   also contributes to tumor cell apoptosis .  
we have previously shown that t - oligo treatment activates ( phosphorylates ) atm     [ 21 , 27 ] .
we therefore suggest that t - oligo regulates e2f1 first via atm - mediated phosphorylation of e2f1      and that e2f1 then further transcriptionally upregulates e2f1 by binding to its own promoter .   indeed
, we show that t - oligo increases e2f1 mrna and protein levels as well as its dna binding activity in human mm - an melanoma cells , further confirming our previous finding that e2f1contributes to t - oligo effects .  
the t - oligos used in the present experiments have physiologic readily hydrolysable phosphodiester linkage , unlike for example antisense dna in which phosphorothioate or other nonhydrolyzable linkage is employed to increase the molecule 's half - life ( t 1/2 ) .  
hydrolyzable linkage is required for initiation of t - oligo signalling   and , despite the known short t 1/2 , approximately 46 hours in serum - containing medium for a 12-base 100% telomere homolog , nevertheless this allows for the cellular responses observed in the present and previous experiments to evolve over 35 days [ 11 , 13 , 19 , 21 , 22 , 2428 ] .  
this may reflect the fact that at least in vitro t - oligos rapidly enter the nucleus [ 11 , 22 , 24 , 27 ] and that , once in the nucleus , such oligos have a far longer t 1/2 .  
the efficacy of these presumptively short - lived t - oligos may also reflect the likelihood that , after interaction of the oligos with the werner protein and formation of dna damage - like foci at telomeres , signalling through the dna damage response pathways may continue even if the t - oligos have been hydrolyzed , at least through 48 hours at which time the dna damage foci can still be observed by immunohistochemistry . in earlier studies , we have shown that t - oligos inhibit tumor growth in scid mouse models by inducing cell cycle arrest , differentiation , apoptosis , and senescence     [ 13 , 15 , 27 ] .  
angiogenesis inhibition encompassing both ecs and melanoma cells , in combination with other t - oligo - mediated anti - tumor effects     [ 11 , 13 , 22 , 25 , 27 ] , likely combine to significantly decrease melanoma burden in established scid mouse models , as reported in our earlier publications     [ 13 , 27 ] .  
these multiple diverse responses are all mediated through activation and/or upregulation of dna damage response proteins .  
they are thus reminiscent of the less complex but very well characterized bacterial sos response , a genome - protective mechanism that enhances survival of prokaryotic organisms in the face of dna damage .  
the telomere - based dna damage - like signalling initiated by t - oligos may be viewed as an evolutionarily perfected parallel mechanism in mammalian cells that addresses the threat to genomic integrity posed by malignant transformation . in summary , the present paper indicates that t - oligos exert multiple antiangiogenic effects .
these data reinforce prior evidence that t - oligo therapy may offer a powerful new approach to treatment of human primary melanoma and possibly other human malignancies , with several conceptual advantages over the currently lionized targeted therapy approach .
 telomere homolog oligonucleotides ( t - oligos ) activate an innate telomere - based program that leads to multiple anticancer effects . 
 t - oligos act at telomeres to initiate signaling through the werner protein and atm kinase . 
 we wanted to determine if t - oligos have antiangiogenic effects . 
 we found that t - oligo - treated human melanoma ( mm - an ) cells had decreased expression of vascular endothelial growth factor ( vegf ) , vegf receptor 2 , angiopoeitin-1 and -2 and decreased vegf secretion . 
 t - oligos activated the transcription factor e2f1 and inhibited the activity of the angiogenic transcription factor , hif-1. t - oligos inhibited ec tubulogenesis and total tumor microvascular density matrix invasion by mm - an cells and ecs in vitro . in melanoma scid xenografts , two systemic t - oligo injections decreased by 60% ( p < .004 ) total tumor microvascular density and the functional vessels density by 80% ( p 
 < .002 ) . 
 these findings suggest that restriction of tumor angiogenesis is among the host 's innate telomere - based anticancer responses and provide further evidence that t - oligos may offer a powerful new approach for melanoma treatment . 