clinical studies have shown that therapeutic agents preventing the synthesis and actions of estrogens are highly successful in the treatment of breast cancer . the mechanisms by which estrogens stimulate cell proliferation , however , are still not clear . the epithelial breast cancer derived mcf-7 cell line is one of the most frequently used model systems . many groups have reported that addition of e2 to the medium of these cells induces a proliferative response [ 1 - 5 ] . however , a comparison of different laboratory strains of mcf-7 reveals that the degree of stimulation differs , and may even be absent [ 6 - 9 ] . recently , we have demonstrated that e2 does not induce cell proliferation in the mcf-7s cell line under serum - free , steroid hormone - free conditions . however , e2 in synergism with submitogenic concentrations of insulin - like growth factor i ( igf - i ; 2 ng / ml ) does induce a proliferative response , comparable to the response to mitogenic amounts of igf - i ( 20 ng / ml ) . we have found that both e2 and mitogenic amounts of igf - i strongly induce cyclin d1 expression , whereas submitogenic amounts of igf - i do not significantly elevate cyclin d1 levels . igf - i , but not e2 , is able to activate pi3-kinase , which leads to inhibition of gsk3 activity . here inhibition of gsk3 triggers nuclear accumulation of the cyclin d1 , but only if cyclin d1 levels are strongly induced concomitantly , which is effectuated by e2 . after cyclin d1 accumulates in the nucleus , activation of the cyclin d1/cdk4 complex and subsequent cell cycle progression is observed . in contrast to other laboratory mcf-7 strains , mcf-7s cells are almost completely growth arrested in g0/g1 phase of the cell cycle by serum deprivation in estrogen - free medium , without the need for estrogen antagonists or other inhibitors to reach quiescence . other laboratory strains of mcf-7 proliferate well when e2 is added after serum - deprivation . in the present study , we compare three distinct mcf-7 strains to determine the cause of their differences in sensitivity for e2 . the mcf-7 cell line from the american type culture collection ( referred to as " mcf-7 atcc " ) shows an intermediate e2-response . the mcf-7 line obtained from the dutch cancer institute in amsterdam , the e2-responsiveness of the three mcf-7 strains was measured and compared to their response to igf - i in a dna - synthesis assay . after 26 h , 1 nm e2 , 20 ng / ml of igf - i ( i(20 ) ) , 2 ng / ml of igf - i ( i(2 ) ) or the combination of 1 nm e2 and 2 ng / ml of igf - i ( i(2)e2 ) were added . twenty - four h later , h - thymidine ( h - tdr ) was added for a 6 h period , after which the cells were harvested . figure 1 shows the h - tdr incorporation in the mcf-7 cell lines , normalized to h - tdr incorporation induced by 20 ng / ml of igf - i . a small amount of h - tdr is incorporated in untreated serum - starved mcf-7s cells , approximately 1% of the incorporation induced by 20 ng / ml of igf - i . the mcf-7 atcc and the mcf-7 nki cells show higher h - tdr incorporation values in untreated serum - starved cells , 6 and 12% of the 20 ng / ml igf - i value , respectively . this indicates that these cells are less well synchronized by serum- and steroid - deprivation and may need additional treatment to reach quiescence . in mcf-7s cells , only a moderate increase in h - tdr incorporation is induced by stimulation with either 2 ng / ml of igf - i or 1 nm e2 in mcf-7s cells , to 3 and 9% of the igf - i ( 20 ng / ml ) value respectively . when both igf - i ( 2 ng / ml ) and e2 are added , a strong increase in h - tdr incorporation is observed to 88% of the incorporation level of 20 ng / ml of igf - i . thus , a clear synergism of e2 and igf - i on the induction of dna - synthesis in mcf-7s cells is demonstrated . in mcf-7 atcc cells , the h - tdr incorporation induced by 2 ng / ml of igf - i is much higher ( 26% of the incorporation seen with 20 ng / ml of igf - i ) than in mcf-7s cells . also 1 nm e2 induces a much larger increase in incorporation ( 40% ) when compared to the incorporation in mcf-7s cells . however , the combination of the two still is synergistic , resulting in a higher incorporation level ( 115% ) than the added effect of the two hormones individually ( 66% ) . in mcf-7 nki cells , e2 is an even stronger inducer of h - tdr incorporation . in this mcf-7 strain , addition of e2 results in a higher incorporation level than observed with mitogenic amounts of igf - i . no synergy between igf - i and e2 can be observed in mcf-7 nki , since h - tdr incorporation induced by 1 nm e2 by itself is as high as by the combination of igf - i ( 2 ng / ml ) and 1 nm e2 . effect of igf - i and e2 on the induction of dna - synthesis in the mcf-7 strains . serum - starved cells grown in 24-wells plates were given no mitogens ( - ) , 20 ng / ml of igf - i ( i(20 ) ) , 2 ng / ml of igf - i ( i(2 ) ) , 2 ng / ml of igf - i in combination with 1 nm e2 ( i(2)e2 ) , or 1 nm e2 ( e2 ) and were harvested after 30 h. h - thymidine ( 2 ci / ml ) was added to the culture 6 h before harvesting . indicated values are means of triplicate measurements , and were normalized for the incorporation induced by 20 ng / ml of igf - i ( 100% ) . to investigate whether mcf-7 atcc and mcf-7 nki express higher levels of er than mcf-7s , we analyzed the er content of serum - starved mcf-7s , mcf-7 atcc , and mcf-7 nki cells by western blotting . we find that the level of er expression in mcf-7s and mcf-7 nki are comparable , whereas the mcf-7 atcc express slightly higher levels of er ( figure 2a ) . the observed difference in expression levels does not correlate with e2 inducible proliferation in the three strains of mcf-7 . b induction of cyclin d1 gene expression in mcf-7s , mcf-7 atcc , and mcf-7 nki by e2 . serum - starved mcf-7s cells were grown in 6-wells plates , and were given no mitogens ( - ) , or 1 nm e2 ( e2 ) . the cells were lysed after 6 h. levels of cyclin d1 were analyzed by western blotting . in order to detect differences in functionality of the er , we analyzed the expression level of an e2-responsive gene , cyclin d1 , in untreated serum - starved and in e2-treated cells . like in mcf-7s cells , e2 treatment induces a strong increase in cyclin d1 levels in both mcf-7 atcc and mcf-7 nki cells . no significant differences in cyclin d1 induction were observed in the three mcf-7 strains ( figure 2b ) . taken together , these results indicate that the differences in growth response upon e2 treatment of the mcf-7 strains are not caused by their er expression level or functionality . to investigate whether e2 can induce cell cycle progression in mcf-7 atcc and nki cells without subsequent activation of the igf type i receptor , a dna synthesis assay was performed in which 200 g / ml suramin was added to the cells one hour prior to stimulation with igf - i , e2 or the combination of both hormones . suramin is an anti - tumor agent inhibiting the proliferation of a variety of human cancer cell lines cultured in vitro [ 12 - 15 ] . suramin acts extracellularly by interfering with the binding of growth factors to their receptors , including several which are known to be involved in tumor growth or development , like igf - i , and tgf . we find that igf - i - induced incorporation of h - tdr is completely inhibited in all three mcf-7 cell lines , when the cells are preincubated with suramin ( figure 3 ) . surprisingly , preincubation with suramin also completely abolishes e2-induced dna synthesis in all three mcf-7 laboratory strains . these results suggest that activation of a suramin - sensitive receptor is essential for e2-induced cell cycle progression . effect of suramin on the induction of dna - synthesis by igf - i and e2 in the three mcf-7 strains . serum - starved cells were grown in 24-wells plates , and were preincubated for 1 hour with 200 g / ml suramin before stimulation with mitogens . cells were given no mitogens ( - ) , 20 ng / ml of igf - i ( i(20 ) ) , 2 ng / ml of igf - i ( i(2 ) ) , 2 ng / ml of igf - i in combination with 1 nm e2 ( i(2)e2 ) , or 1 nm e2 ( e2 ) and were harvested after 30 h. h - thymidine ( 2 ci / ml ) was added to the culture 6 h before harvesting . the values were normalized for the incorporation induced by 20 ng / ml of igf - i ( 100% ) . to test whether e2-responsiveness is dependent on igf - signaling , the igf - ri was blocked by the addition of 1 g / ml ir3 antibody to the culture medium , which prevents binding of igf - i and -ii to the receptor . figure 4 shows that igf - i - induced h - tdr incorporation is completely inhibited in all three strains of mcf-7 treated with ir3 . however , also the effect of e2 on h - tdr incorporation is reduced to levels comparable to the incorporation in untreated serum - starved cells . this shows that all three mcf-7 strains loose their e2-responsiveness when igf - ri signaling is blocked . effect of an igf - ri blocking antibody on the induction of dna - synthesis by igf - i and e2 in the three mcf-7 strains . serum - starved cells were grown in 24-wells plates , and were preincubated for 1 hour with 1 g / ml of ir3 antibody before stimulation with mitogens . cells were treated with no mitogens ( - ) , 20 ng / ml of igf - i ( i(20 ) ) , 2 ng / ml of igf - i ( i(2 ) ) , 2 ng / ml of igf - i in combination with 1 nm e2 ( i(2)e2 ) , or 1 nm e2 ( e2 ) and were harvested after 30 h. h - thymidine ( 2 ci / ml ) was added to the culture 6 h before harvesting . indicated values are means of triplicate measurements . the values were normalized for the incorporation induced by 20 ng / ml of igf - i ( 100% ) . the observed inhibition of dna synthesis theoretically might be due to toxicity of the antibodies added to the cells . to exclude this possibility , the specificity of inhibition was examined by determining whether other signaling pathways are still intact in antibody treated cells . to this end , tpa induces the synthesis of p21via activation of the erk pathway independent of igf - ri . a moderate elevation of p21levels is also observed when mcf-7 cells are treated with mitogenic amounts of igf - i . figure 5 shows that the addition of 100 ng / ml of tpa to the medium induces expression of p21 . igf - i ( 20 ng / ml ) also clearly results in higher p21levels . ir3 is able to totally block the upregulation of p21induced by igf - i , but does not inhibit the stimulation of p21expression by tpa . these results show that the inhibition of e2-induced dna synthesis in e2-responsive mcf-7 cells by ir3 is a result of specific inhibition of igf - ri signaling . effect of an igf - ri blocking antibody on the induction of p21 serum - starved mcf-7s cells were grown in 6-wells plates , and were preincubated for 1 hour with 1 g / ml of ir3 antibody before stimulation with mitogens . they were given no mitogens ( - ) , 20 ng / ml of igf - i ( i(20 ) ) , or 100 ng / ml of tpa ( tpa ) and were lysed after 4 h. levels of p21were analyzed by western blotting . in view of the fact that activation of the igf - ri is important for e2-induced cell cycle progression in mcf-7 atcc and mcf-7 nki cells , we investigated the possibility of autocrine stimulation of the igf - ri in these mcf-7 strains . to this end , we studied the effect of conditioned media on the e2-responsiveness of the mcf-7 strains in a dna - synthesis assay . mcf-7s , atcc and nki cells were seeded in 162 cmculture flasks and incubated in serum - free , estrogen - free medium for two days , after which the conditioned media were collected . for the dna - synthesis assay , mcf-7s , mcf-7 atcc and mcf-7 nki cells were seeded in 24 wells plates , and serum - starved for 24 h. prior to stimulation the serum - free medium on the three mcf-7 strains was replaced by the conditioned media , or by fresh serum - free medium as a control . after 15 minutes , no mitogens , igf - i ( 2 or 20 ng / ml ) , 1 nm e2 or the combination of 1 nm e2 and 2 ng / ml igf - i were added to the media . 24 h later , h - tdr was added for a 6 h period , after which the cells were harvested . figure 6 shows the effect of the conditioned media on the mcf-7s cells . in untreated steroid- and serum - free medium , mcf-7s cells culturing the cells in conditioned medium from the mcf-7s cell line has no significant effect on the h - tdr incorporation induced by any of the hormone treatments . however , when e2 is administered together with conditioned medium from mcf-7 atcc or mcf-7 nki , the cells progress through s - phase as is demonstrated by elevated incorporation of h - tdr in these cells . effect of conditioned serum - free media from mcf-7 cells on the induction of dna - synthesis by igf - i and e2 in the three mcf-7 strains . conditioned media ( cm ) were generated by incubating mcf-7s , atcc and nki cells in serum - free , estrogen - free medium for two days , after which the medium was collected and centrifuged to spin down the cell debris . serum - starved cells were grown in 24-wells plates , and were incubated in normal serum - free medium ( indicated as [ - ] in the figure legend ) or in the conditioned serum - free media ( indicated as cm mcf-7s , cm mcf-7atcc , or cm mcf-7 nki ) . the cells were given no mitogens ( - ) , 20 ng / ml of igf - i ( i(20 ) ) , 2 ng / ml of igf - i ( i(2 ) ) , 2 ng / ml of igf - i in combination with 1 nm e2 ( i(2)e2 ) , or 1 nm e2 ( e2 ) and were harvested after 30 h. h - thymidine ( 2 ci / ml ) was added to the culture 6 h before harvesting . the values were normalized for the incorporation induced by 20 ng / ml of igf - i ( 100% ) . also in the mcf-7 atcc cell line , the conditioned media from mcf-7 atcc and mcf-7 nki , but not the conditioned medium of mcf-7s , induce additional h - tdr incorporation in cells treated with e2 when compared to cells incubated in fresh serum - free medium with e2 . in mcf-7 atcc , incubation in the conditioned media from the two e2-inducible cell lines leads to a higher incorporation in untreated and in igf - i ( 2 ng / ml ) treated cells . this increase is less pronounced in the mcf-7s cell line ( figure 6 ) . in mcf-7 nki cells , the conditioned media have no significant effect on the amount of incorporation induced by e2 , but they do stimulate h - tdr incorporation in untreated and in igf - i ( 2 ng / ml ) stimulated cells ( figure 6 ) . these results suggest that the mcf-7 atcc and mcf-7 nki cells secrete a factor into the medium that activates the igf - ri and synergizes with e2 to induce cell cycle progression in all three strains of mcf-7 . the e2-responsiveness of the three mcf-7 strains was measured and compared to their response to igf - i in a dna - synthesis assay . after 26 h , 1 nm e2 , 20 ng / ml of igf - i ( i(20 ) ) , 2 ng / ml of igf - i ( i(2 ) ) or the combination of 1 nm e2 and 2 ng / ml of igf - i ( i(2)e2 ) were added . twenty - four h later , h - thymidine ( h - tdr ) was added for a 6 h period , after which the cells were harvested . figure 1 shows the h - tdr incorporation in the mcf-7 cell lines , normalized to h - tdr incorporation induced by 20 ng / ml of igf - i . a small amount of h - tdr is incorporated in untreated serum - starved mcf-7s cells , approximately 1% of the incorporation induced by 20 ng / ml of igf - i . the mcf-7 atcc and the mcf-7 nki cells show higher h - tdr incorporation values in untreated serum - starved cells , 6 and 12% of the 20 ng / ml igf - i value , respectively . this indicates that these cells are less well synchronized by serum- and steroid - deprivation and may need additional treatment to reach quiescence . in mcf-7s cells , only a moderate increase in h - tdr incorporation is induced by stimulation with either 2 ng / ml of igf - i or 1 nm e2 in mcf-7s cells , to 3 and 9% of the igf - i ( 20 ng / ml ) value respectively . when both igf - i ( 2 ng / ml ) and e2 are added , a strong increase in h - tdr incorporation is observed to 88% of the incorporation level of 20 ng / ml of igf - i . thus , a clear synergism of e2 and igf - i on the induction of dna - synthesis in mcf-7s cells is demonstrated . in mcf-7 atcc cells , the h - tdr incorporation induced by 2 ng / ml of igf - i is much higher ( 26% of the incorporation seen with 20 ng / ml of igf - i ) than in mcf-7s cells . also 1 nm e2 induces a much larger increase in incorporation ( 40% ) when compared to the incorporation in mcf-7s cells . however , the combination of the two still is synergistic , resulting in a higher incorporation level ( 115% ) than the added effect of the two hormones individually ( 66% ) . in mcf-7 nki cells , e2 is an even stronger inducer of h - tdr incorporation . in this mcf-7 strain , addition of e2 results in a higher incorporation level than observed with mitogenic amounts of igf - i . no synergy between igf - i and e2 can be observed in mcf-7 nki , since h - tdr incorporation induced by 1 nm e2 by itself is as high as by the combination of igf - i ( 2 ng / ml ) and 1 nm e2 . effect of igf - i and e2 on the induction of dna - synthesis in the mcf-7 strains . serum - starved cells grown in 24-wells plates were given no mitogens ( - ) , 20 ng / ml of igf - i ( i(20 ) ) , 2 ng / ml of igf - i ( i(2 ) ) , 2 ng / ml of igf - i in combination with 1 nm e2 ( i(2)e2 ) , or 1 nm e2 ( e2 ) and were harvested after 30 h. h - thymidine ( 2 ci / ml ) was added to the culture 6 h before harvesting . indicated values are means of triplicate measurements , and were normalized for the incorporation induced by 20 ng / ml of igf - i ( 100% ) . to investigate whether mcf-7 atcc and mcf-7 nki express higher levels of er than mcf-7s , we analyzed the er content of serum - starved mcf-7s , mcf-7 atcc , and mcf-7 nki cells by western blotting . we find that the level of er expression in mcf-7s and mcf-7 nki are comparable , whereas the mcf-7 atcc express slightly higher levels of er ( figure 2a ) . the observed difference in expression levels does not correlate with e2 inducible proliferation in the three strains of mcf-7 . b induction of cyclin d1 gene expression in mcf-7s , mcf-7 atcc , and mcf-7 nki by e2 . serum - starved mcf-7s cells were grown in 6-wells plates , and were given no mitogens ( - ) , or 1 nm e2 ( e2 ) . the cells were lysed after 6 h. levels of cyclin d1 were analyzed by western blotting . in order to detect differences in functionality of the er , we analyzed the expression level of an e2-responsive gene , cyclin d1 , in untreated serum - starved and in e2-treated cells . like in mcf-7s cells , e2 treatment induces a strong increase in cyclin d1 levels in both mcf-7 atcc and mcf-7 nki cells . no significant differences in cyclin d1 induction were observed in the three mcf-7 strains ( figure 2b ) . taken together , these results indicate that the differences in growth response upon e2 treatment of the mcf-7 strains are not caused by their er expression level or functionality . to investigate whether e2 can induce cell cycle progression in mcf-7 atcc and nki cells without subsequent activation of the igf type i receptor , a dna synthesis assay was performed in which 200 g / ml suramin was added to the cells one hour prior to stimulation with igf - i , e2 or the combination of both hormones . suramin is an anti - tumor agent inhibiting the proliferation of a variety of human cancer cell lines cultured in vitro [ 12 - 15 ] . suramin acts extracellularly by interfering with the binding of growth factors to their receptors , including several which are known to be involved in tumor growth or development , like igf - i , and tgf . we find that igf - i - induced incorporation of h - tdr is completely inhibited in all three mcf-7 cell lines , when the cells are preincubated with suramin ( figure 3 ) . surprisingly , preincubation with suramin also completely abolishes e2-induced dna synthesis in all three mcf-7 laboratory strains . these results suggest that activation of a suramin - sensitive receptor is essential for e2-induced cell cycle progression . effect of suramin on the induction of dna - synthesis by igf - i and e2 in the three mcf-7 strains . serum - starved cells were grown in 24-wells plates , and were preincubated for 1 hour with 200 g / ml suramin before stimulation with mitogens . cells were given no mitogens ( - ) , 20 ng / ml of igf - i ( i(20 ) ) , 2 ng / ml of igf - i ( i(2 ) ) , 2 ng / ml of igf - i in combination with 1 nm e2 ( i(2)e2 ) , or 1 nm e2 ( e2 ) and were harvested after 30 h. h - thymidine ( 2 ci / ml ) was added to the culture 6 h before harvesting . the values were normalized for the incorporation induced by 20 ng / ml of igf - i ( 100% ) . to test whether e2-responsiveness is dependent on igf - signaling , the igf - ri was blocked by the addition of 1 g / ml ir3 antibody to the culture medium , which prevents binding of igf - i and -ii to the receptor . figure 4 shows that igf - i - induced h - tdr incorporation is completely inhibited in all three strains of mcf-7 treated with ir3 . however , also the effect of e2 on h - tdr incorporation is reduced to levels comparable to the incorporation in untreated serum - starved cells . this shows that all three mcf-7 strains loose their e2-responsiveness when igf - ri signaling is blocked . effect of an igf - ri blocking antibody on the induction of dna - synthesis by igf - i and e2 in the three mcf-7 strains . serum - starved cells were grown in 24-wells plates , and were preincubated for 1 hour with 1 g / ml of ir3 antibody before stimulation with mitogens . cells were treated with no mitogens ( - ) , 20 ng / ml of igf - i ( i(20 ) ) , 2 ng / ml of igf - i ( i(2 ) ) , 2 ng / ml of igf - i in combination with 1 nm e2 ( i(2)e2 ) , or 1 nm e2 ( e2 ) and were harvested after 30 h. h - thymidine ( 2 ci / ml ) was added to the culture 6 h before harvesting . the values were normalized for the incorporation induced by 20 ng / ml of igf - i ( 100% ) . the observed inhibition of dna synthesis theoretically might be due to toxicity of the antibodies added to the cells . to exclude this possibility , the specificity of inhibition was examined by determining whether other signaling pathways are still intact in antibody treated cells . to this end , the tpa - induced synthesis of the cki p21protein was studied on western blot . tpa induces the synthesis of p21via activation of the erk pathway independent of igf - ri . a moderate elevation of p21levels is also observed when mcf-7 cells are treated with mitogenic amounts of igf - i . figure 5 shows that the addition of 100 ng / ml of tpa to the medium induces expression of p21 . igf - i ( 20 ng / ml ) also clearly results in higher p21levels . ir3 is able to totally block the upregulation of p21induced by igf - i , but does not inhibit the stimulation of p21expression by tpa . these results show that the inhibition of e2-induced dna synthesis in e2-responsive mcf-7 cells by ir3 is a result of specific inhibition of igf - ri signaling . effect of an igf - ri blocking antibody on the induction of p21 serum - starved mcf-7s cells were grown in 6-wells plates , and were preincubated for 1 hour with 1 g / ml of ir3 antibody before stimulation with mitogens . they were given no mitogens ( - ) , 20 ng / ml of igf - i ( i(20 ) ) , or 100 ng / ml of tpa ( tpa ) and were lysed after 4 h. levels of p21were analyzed by western blotting . in view of the fact that activation of the igf - ri is important for e2-induced cell cycle progression in mcf-7 atcc and mcf-7 nki cells , we investigated the possibility of autocrine stimulation of the igf - ri in these mcf-7 strains . to this end , we studied the effect of conditioned media on the e2-responsiveness of the mcf-7 strains in a dna - synthesis assay . mcf-7s , atcc and nki cells were seeded in 162 cmculture flasks and incubated in serum - free , estrogen - free medium for two days , after which the conditioned media were collected . for the dna - synthesis assay , mcf-7s , mcf-7 atcc and mcf-7 nki cells were seeded in 24 wells plates , and serum - starved for 24 h. prior to stimulation the serum - free medium on the three mcf-7 strains was replaced by the conditioned media , or by fresh serum - free medium as a control . after 15 minutes , no mitogens , igf - i ( 2 or 20 ng / ml ) , 1 nm e2 or the combination of 1 nm e2 and 2 ng / ml igf - i were added to the media . 24 h later , h - tdr was added for a 6 h period , after which the cells were harvested . figure 6 shows the effect of the conditioned media on the mcf-7s cells . in untreated steroid- and serum - free medium , mcf-7s cells culturing the cells in conditioned medium from the mcf-7s cell line has no significant effect on the h - tdr incorporation induced by any of the hormone treatments . however , when e2 is administered together with conditioned medium from mcf-7 atcc or mcf-7 nki , the cells progress through s - phase as is demonstrated by elevated incorporation of h - tdr in these cells . effect of conditioned serum - free media from mcf-7 cells on the induction of dna - synthesis by igf - i and e2 in the three mcf-7 strains . conditioned media ( cm ) were generated by incubating mcf-7s , atcc and nki cells in serum - free , estrogen - free medium for two days , after which the medium was collected and centrifuged to spin down the cell debris . serum - starved cells were grown in 24-wells plates , and were incubated in normal serum - free medium ( indicated as [ - ] in the figure legend ) or in the conditioned serum - free media ( indicated as cm mcf-7s , cm mcf-7atcc , or cm mcf-7 nki ) . the cells were given no mitogens ( - ) , 20 ng / ml of igf - i ( i(20 ) ) , 2 ng / ml of igf - i ( i(2 ) ) , 2 ng / ml of igf - i in combination with 1 nm e2 ( i(2)e2 ) , or 1 nm e2 ( e2 ) and were harvested after 30 h. h - thymidine ( 2 ci / ml ) was added to the culture 6 h before harvesting . the values were normalized for the incorporation induced by 20 ng / ml of igf - i ( 100% ) . also in the mcf-7 atcc cell line , the conditioned media from mcf-7 atcc and mcf-7 nki , but not the conditioned medium of mcf-7s , induce additional h - tdr incorporation in cells treated with e2 when compared to cells incubated in fresh serum - free medium with e2 . in mcf-7 atcc , incubation in the conditioned media from the two e2-inducible cell lines leads to a higher incorporation in untreated and in igf - i ( 2 ng / ml ) treated cells . this increase is less pronounced in the mcf-7s cell line ( figure 6 ) . in mcf-7 nki cells , the conditioned media have no significant effect on the amount of incorporation induced by e2 , but they do stimulate h - tdr incorporation in untreated and in igf - i ( 2 ng / ml ) stimulated cells ( figure 6 ) . these results suggest that the mcf-7 atcc and mcf-7 nki cells secrete a factor into the medium that activates the igf - ri and synergizes with e2 to induce cell cycle progression in all three strains of mcf-7 . mcf-7s cells do not proliferate upon addition of only e2 ( 1 nm ) to serum - free medium . other strains of mcf-7 , here indicated as mcf-7 atcc and mcf-7 nki , which were tested under identical conditions as mcf-7s , do respond to e2-treatment . we have shown that that the observed differences in growth response upon e2-treatment of the mcf-7 strains are not caused by differences in er expression level or functionality . the effect of e2 on the incorporation of h - tdr is completely inhibited by suramin - preincubation in all three cell lines to levels lower than background incorporation in untreated cells . more specifically , we have shown that the induction of dna - synthesis by e2 in all three cell lines could be blocked completely by incubating the cells with 1 g / ml of ir3 antibody , which binds to the igf - ri and thereby inhibits the binding of ligand . from this we conclude that e2-induced dna synthesis in mcf-7 cells is dependent on igf - ri signaling . our results indicate that the e2-responsive strains , mcf-7 atcc and mcf-7 nki , autonomously activate their igf - ir , and that this activation is required for their response to e2 . since endogenous production of growth factors is a common feature of many tumor cells , we investigated whether the two e2-responsive mcf-7 strains secrete an autocrine factor capable of activating their igf - ri . we demonstrated that e2-insensitive cells become e2-sensitive when challenged with conditioned medium of e2-sensitive cells . this suggests that e2-sensitive cells secrete an autocrine factor capable of activating the igf - ri . differences in the levels of production of igf - ir agonists by the breast cancer cells may explain the differences observed in the e2-sensitivity of the three mcf-7 strains . van der burg et al . , using a sensitive bioassay , could not detect any igf - i or -ii production in mcf-7s cells . other reports indicate expression of igfs by certain mcf-7 strains . the majority of reports studying this issue report the absence of detectable igf - i expression in mcf-7 . expression of igf - ii mrna has been detected by northern blot analysis and rnase protection assays . after removal of the binding proteins , concentrations between 4.7 and 14.4 ng / ml per 10mcf-7 cells of igf - ii protein has been found in conditioned medium depending on the technique used ( ria and radioreceptor assays ) . significantly , yee et al . observed that igf - ii expression by mcf-7 cells increases with the passage number of the cell culture . this observation suggests that the factor secreted by e2-sensitive cells may be igf - ii . igf - ii has been shown to induce a mitogenic response in a variety of breast cancer cell lines . it has been found to be slightly less potent than igf - i , since 24 times higher concentrations igf - ii are needed for equal responses in a dna synthesis assay . even so , the concentrations reportedly present in the conditioned medium of mcf-7 cells   are sufficient to synergistically induce a mitogenic response in mcf-7 cells upon addition of e2 . the reason for the change in igf - ii expression over time is not clear . the mcf-7 cell line was isolated from a pleural effusion derived from a breast carcinoma , and was shown to consist of several different subpopulations of cells . it is plausible that during cultivation of the initially heterogeneous cell line an igf - ii - expressing cell type is naturally selected because of its growth advantage over non - growth - factor expressing cells . mcf-7s cells , in contrast to other laboratory mcf-7 strains , are almost completely growth arrested in g0/g1 phase of the cell cycle by serum deprivation in estrogen - free medium , without the need for estrogen antagonists or other inhibitors to reach quiescence . we have found that the mcf-7s cells show this characteristic for about 25 to 30 passages , after which the cells start to grow more rapidly and do no longer show complete growth arrest upon serum withdrawal . furthermore , during cell culture the cells become more igf independent and significant proliferation can be induced by the sole addition of e2 to the serum - free medium . endogenous expression of igf - i or igf - ii by e2-sensitive mcf-7 strains could account for their observed e2 inducible growth , independent of igf - i addition . the change in e2-responsiveness of mcf-7s may indicate that prolonged growth in tissue culture triggers the production of igf . alternatively , igf - producing cells that were already present in the original culture gain a growth advantage and gradually overgrow the non - igf - producing cells . in conclusion , the differential response of different mcf-7 strains to e2 and igf - i may well be due to differences in the individual igf - expression status in these strains . using rt - pcr , we found than none of the mcf-7 strains produce igf - i . however , in all three of the cell lines igf - ii mrna could be detected ( data not shown ) . thus , igf - ii may well be the autocrine factor involved in regulation of the response to e2 . in conclusion , we have shown that e2-responsiveness of mcf-7 cells is dependent on the secretion of an autocrine factor activating the igf - ir . all three strains of mcf-7 breast cancer cells investigated do not respond to e2 if the igf - ri - pathway is blocked . this study suggests that inhibition of igf - action in combination with anti - estrogen - treatment may provide a more effective way in treatment or even prevention of breast cancer . anti - p21(h-164 ) was purchased from santa cruz biotechnology ( santa cruz , ca ) . aprotinin , e2 , sodium selenite , suramin , and tpa were purchased from sigma ( st . louis , mo ) . albumin , bovine fraction v ( bsa , catalogue number 840533 ) , na3vo4 and transferrin were from icn biomedicals , inc . s. van buul - offers from the department of pediatric endocrinology , umc utrecht , the netherlands . the mcf-7 human breast cancer cell lines were cultured in dulbecco 's minimal essential medium / ham 's f12 medium ( 1:1 ) , supplemented with 10% fetal calf serum , 300 g / ml of glutamine , 100 iu / ml of penicillin and 100 g / ml of streptomycin . mcf-7 cells were cultured for 24 h in phenol red - free medium containing 5% dextran - coated charcoal - treated serum , followed by an incubation in phenol red - free , serum - free medium , supplemented with 0.2% bovine serum albumin ( bsa ) , 10 g / ml of transferrin and 30 nm sodium selenite for at least 24 h. assays were performed in 24-well plates . serum - starved cells were stimulated with igf - i , e2 , or a combination of the two and after 24 h of incubation h - labeled thymidine ( 2 ci / ml ) was added . 30 h after the addition of the stimuli , cells were fixed with 10% trichloroacetic acid , washed in pbs and lysed in 0.1 m naoh , followed by liquid scintillation counting . after stimulation , cells were washed with ice - cold pbs and lysed in lysis buffer ( 50 mm tris - hcl , ph 6.8 , 2% sds , 10% glycerol ) . lysates were boiled for 5 min and protein concentrations were determined using the bca protein assay ( pierce , rockford , il ) . after addition of 0.1% bromophenol blue and 5% -mercaptoethanol to the samples , equal amounts of protein ( 550 g ) were size - separated on a sds - polyacrylamide gel and transferred to nitrocellulose . the membrane was stained with ponceau s ( sigma ) to check for equal loading and blocked with 23% non - fat milk or 0.52% bsa in tbst ( 150 mm nacl , 10 mm tris - hcl ph 8.0 , 0.2% tween 20 ) for 1 h. incubation with the primary antibody was performed overnight at 4c in 0.1  blocking solution . the membrane was washed in tbst and exposed to horseradish peroxidase - coupled secondary antibody for 1 h in tbst . for detection the western blot chemiluminescence reagent plus system ( nen life science products inc . anti - p21(h-164 ) was purchased from santa cruz biotechnology ( santa cruz , ca ) . aprotinin , e2 , sodium selenite , suramin , and tpa were purchased from sigma ( st . louis , mo ) . albumin , bovine fraction v ( bsa , catalogue number 840533 ) , na3vo4 and transferrin were from icn biomedicals , inc . s. van buul - offers from the department of pediatric endocrinology , umc utrecht , the netherlands . the mcf-7 human breast cancer cell lines were cultured in dulbecco 's minimal essential medium / ham 's f12 medium ( 1:1 ) , supplemented with 10% fetal calf serum , 300 g / ml of glutamine , 100 iu / ml of penicillin and 100 g / ml of streptomycin . mcf-7 cells were cultured for 24 h in phenol red - free medium containing 5% dextran - coated charcoal - treated serum , followed by an incubation in phenol red - free , serum - free medium , supplemented with 0.2% bovine serum albumin ( bsa ) , 10 g / ml of transferrin and 30 nm sodium selenite for at least 24 h. serum - starved cells were stimulated with igf - i , e2 , or a combination of the two and after 24 h of incubation h - labeled thymidine ( 2 ci / ml ) was added . 30 h after the addition of the stimuli , cells were fixed with 10% trichloroacetic acid , washed in pbs and lysed in 0.1 m naoh , followed by liquid scintillation counting . experiments were performed in 6-well plates . after stimulation , cells were washed with ice - cold pbs and lysed in lysis buffer ( 50 mm tris - hcl , ph 6.8 , 2% sds , 10% glycerol ) . lysates were boiled for 5 min and protein concentrations were determined using the bca protein assay ( pierce , rockford , il ) . after addition of 0.1% bromophenol blue and 5% -mercaptoethanol to the samples , equal amounts of protein ( 550 g ) were size - separated on a sds - polyacrylamide gel and transferred to nitrocellulose . the membrane was stained with ponceau s ( sigma ) to check for equal loading and blocked with 23% non - fat milk or 0.52% bsa in tbst ( 150 mm nacl , 10 mm tris - hcl ph 8.0 , 0.2% tween 20 ) for 1 h. incubation with the primary antibody was performed overnight at 4c in 0.1  blocking solution . the membrane was washed in tbst and exposed to horseradish peroxidase - coupled secondary antibody for 1 h in tbst . for detection the western blot chemiluminescence reagent plus system ( nen life science products inc . , ih carried out the western blots , the dna synthesis assays and drafted the article . e2 , 17-estradiol ; er , estrogen receptor ; igf , insulin - like growth factor ; igf - bp , igf binding protein ; igf - ri , insulin - like growth factor type i receptor ; mcf-7 , michigan cancer foundation 7 cell line , derived from an epithelial breast cancer ; tpa , 12-o - tetradecanoyl phorbol 13-acetate we would like to thank dr . s van buul - offers for the kind gift of ir3 anti - igf - ri antibody , and b. hendriks - stegeman and dr . this work was supported by the dutch cancer foundation , kwf , grant uu 98 - 1701 .
backgroundhuman mcf-7 cells have been studied extensively as a model for breast cancer cell growth . 
 many reports have established that serum - starved mcf-7 cells can be induced to proliferate upon the sole addition of 17-estradiol ( e2 ) . 
 however , the extent of the mitogenic response to e2 varies in different mcf-7 strains and may even be absent . in this study 
 we compared the e2-sensitivity of three mcf-7 laboratory strains.resultsthe mcf-7s line is non - responsive to e2 , the mcf-7 atcc has an intermediate response to e2 , while the mcf-7 nki is highly e2-sensitive , although the levels and activities of the estrogen receptor ( er ) are not significantly different . 
 both suramin and igf type i receptor blocking antibodies are able to inhibit the mitogenic response to e2-treatment in mcf-7 atcc and mcf-7 nki cells . from this 
 we conclude that e2-induced proliferation is dependent on igf type i receptor activation in all three mcf-7 strains.conclusionsthe results presented in this article suggest that e2-responsiveness of mcf-7 cells is dependent on the secretion of an autocrine factor activating the igf - ir . 
 all three strains of mcf-7 breast cancer cells investigated do not respond to e2 if the igf - ri - pathway is blocked . 
 generally , breast cancer therapy is targeted at inhibiting estrogen action . 
 this study suggests that inhibition of igf - action in combination with anti - estrogen - treatment may provide a more effective way in treatment or even prevention of breast cancer .