Expression of Insulin-Like Growth FactorReceptor, IGF-1, and IGF-2 in Primary and

Metastatic Osteosarcoma

SARAH BURROW, MD, MSc,1 IRENE L. ANDRULIS, PhD,1 MICHAEL POLLAK, MD, FRCPC,2 AND

ROBERT S. BELL, MD, FRCSC1*1Samuel Lunenfeld Research Institute, Mount Sinai Hospital and the University of Toronto,

Toronto, Ontario, Canada2Lady Davis Research Institute of the Jewish General Hospital, McGill University,

Montreal, Quebec, Canada

Background and Objectives:We have previously shown that insulin-likegrowth factor (IGF)-responsive murine sarcomas demonstrate inhibitionof local and metastatic disease growth when implanted in an IGF-deficienthost animal. In this experiment, we tested whether IGF receptor (IGF-R)and ligands were expressed in human primary and metastatic osteosarco-mas.Methods: Fifty-two specimens of human osteosarcoma tumor from 48patients were assayed for IGF-R, IGF-1, and IGF-2 using reverse tran-scriptase polymerase chain reaction.Results: Twenty-one of 46 tumors analyzed had levels of expression ofIGF-R greater than or equal to the positive control cell line. Twenty-sevenof 44 expressed levels of IGF-1 greater than or equal to the positivecontrol, as did 21 of 38 cases assayed for IGF-2. No differences werefound between 40 primary tumor samples and 12 metastatic lesions inmean levels of IGF-R, IGF-1, or IGF-2. There was a moderately strongcorrelation between expression of IGF-R and IGF-1, suggesting that au-tocrine stimulation may be an important mechanism for stimulation ofosteosarcoma proliferation.Conclusions: A significant proportion of osteosarcoma tumors expressIGF-R and ligands. Higher levels of expression were not correlated withmetastatic lesions.J. Surg. Oncol. 1998;69:21–27. © 1998 Wiley-Liss, Inc.

KEY WORDS: osteosarcoma; insulin-like growth factor; sarcoma;insulin-like growth factor receptor; bone neoplasms

INTRODUCTION

Despite the use of multidrug adjuvant chemotherapyprotocols, a significant proportion of patients with osteo-sarcoma develop metastatic disease either during or,more commonly, after the completion of drug treatment[1]. Once diagnosed with metastases, the potential forcure in osteosarcoma is markedly decreased [1]. Becausemost patients developing relapse have already receivedmaximal chemotherapy treatment, it is important to con-sider new therapeutic approaches for osteosarcoma thatdo not rely on cytotoxic drugs. Understanding the factorsthat stimulate osteosarcoma growth and metastasis may

be important for developing these novel treatment pro-tocols.

It is evident that insulin like growth factor (IGF) isimportant for the proliferation of some human malignan-

Grant sponsors: the National Cancer Institute of Canada and the Ca-nadian Cancer Society.*Correspondence to: R. Bell, MD, University Musculoskeletal Oncol-ogy Unit, Suite 476, Mount Sinai Hospital, 600 University Avenue,Toronto, Ontario, Canada, M5G 1X5. Fax No.: (416) 586-5397.E-mail: bell@mshri.on.caAccepted 8 June 1998

Journal of Surgical Oncology 1998;69:21–27

© 1998 Wiley-Liss, Inc.

cies including sarcomas [2–5]. The IGF signal transduc-tion system involves ligands, receptors, and binding pro-teins [6–9] and has been shown to be mitogenic for ma-lignant cell lines of both epithelial and mesenchymalorigin [10–15]. IGF-1 is a small peptide that mediates thephysiological effects of growth hormone (GH) on skel-etal growth during the adolescent growth spurt [16,17].IGF-2 has an important physiological role in fetal andneonatal-natal growth, but is rarely expressed in adulttissues [8]. Both of these ligands exert their effect on cellproliferation and differentiation by binding to the IGF-1receptor [18], which is an important co-factor in thetransformation of cells to a malignant phenotype [2].

It has been shown that nontransformed mesenchymalcell lines will grow in serum-free medium when trans-fected with IGF-receptor (IGF-R) DNA [19]. Other in-vestigations using cell lines derived from mouse embryoswith a targeted disruption in the IGF-R gene have shownthat these cells are resistant to transformation with theSV40 T antigen, papillomavirus, or an amplified platelet-derived growth factor receptor [20–22]. A series of ex-periments using antisense strategies against IGF-R RNAhave shown inhibition of soft agar growth in a variety ofhuman cancer cell lines [23,24]. In addition to the effectof IGF signaling in the growth of transformed cells, it hasbeen shown that IGF-2 stimulates motility in sarcomacells in vitro [10].

We have previously shown that a proportion of humansarcomas express IGF-R and IGF-1 and IGF-2 ligands[25] and that sarcoma cell lines are responsive to IGF invitro [14]. We have observed using IGF-responsive mu-rine tumors in vivo that both local tumor growth andmetastasis can be decreased by lowering serum levels ofIGF using surgical hypophysectomy [26,27] or by phar-maceutical agents that block GH production by the pitu-itary [28]. Furthermore, we have demonstrated that theeffect of hypophysectomy on metastasis in IGF-responsive murine tumors can be partially reversed bythe administration of human GH to the hypophysecto-mized host [27]. In similar experiments we have demon-strated that the growth of IGF-responsive murine breasttumors can be inhibited by implantation in mice that arehomozygous for lit, a missense gene mutation resultingin loss of function of the pituitary GH-releasing hormonereceptor and subsequent suppression of serum IGF-1 lev-els [29]. These results suggest that some human sarcomasare responsive to IGF mitogenic signals and that alter-ation of IGF physiology by a variety of therapeutic in-terventions may offer a low-morbidity method for inhi-bition of sarcoma metastatic potential in the clinical set-ting.

In light of these observations, we have questionedwhat proportion of human osteosarcomas express bio-logically significant levels of IGF ligand and IGF-1 re-ceptor and whether metastatic osteosarcoma tumors ex-

press higher levels of ligand or receptor than primarytumors. The experiments reported below confirm ouroriginal finding that both ligands and receptor are pro-duced by a high proportion of human osteosarcomas.However, expression was not increased in samples ob-tained from lung metastases when compared withsamples obtained from primary tumors.

MATERIALS AND METHODSTumor Specimens

Samples from surgically resected primary and meta-static osteosarcoma were obtained from viable regions ofthe tumor and immediately flash frozen in liquid nitro-gen. Frozen sections taken from adjacent regions of thetumor were used to confirm the presence of viable tumor.Specimens were obtained from 48 patients in total. Infour patients samples from both the primary tumor andthe subsequent metastasis were available for analysis. In36 cases, sample was available only from the primarytumor, and in 8 cases material was available only fromthe metastatic lesion.

Control Cell Lines

The MCF-7 breast cancer line was used as a positivecontrol for IGF-R since this cell line is responsive toIGF-1 in vitro and demonstrates inhibited growth whenimplanted in the scid/lit murine host deficient for GH andIGF [29,30]. RPMI 7666 and NCI H69 cell lines wereused as positive controls for IGF-1 and IGF-2, respec-tively, since they have been shown in vitro to producephysiological levels of ligand in conditioned medium[31]. MCF-7 and NCI H69 were maintained in modifiedEagle’s medium (GIBCO, Grand Island, NY) supple-mented with 10% fetal calf serum (FCS), and RPMI 7666was maintained in RPMI 1640 and 20% FCS.

RNA Extraction From Specimens and Cell Lines

A portion of each specimen was crushed in an homog-enizer while frozen and placed in guanidium thiocyanate.RNA was extracted by ultracentrifugation of nucleic acidthrough a CsCl gradient as previously described [25].Cell line RNA was obtained after suspension of mono-layers in guanidium thiocyanate. After removal of pro-tein by phenol-chloroform extraction, the sample wasethanol precipitated and resuspended in diethyl pyrocar-bonate-treated water.

Quantification of RT-PCR Products

Primers specific for the IGF-1 receptor, IGF-1, andIGF-2 have been described previously [25]. The senseIGF-R R1 primer (58-ACCCGGAGTACTTCAGCGCT-38) corresponded to nucleotides 2,975–2,994 in exon 14.The antisense primer R2 (58-CACAGAAGCTTCGTT-GAGAA-38) corresponded to nucleotides 3,185–3,204 inexon 16.

22 Burrow et al.

The sense primer for IGF-1, IG1 (58-TCTTGA-AGGTGAAGATGCACACCA-38), corresponded tonucleotides 335–342 in exon 1 and 1–6 in exon 2. Theanti-sense primer IG2 (58-AGCGAGCTGACTTG-GCAGGCTTGA-38) corresponded to nucleotides 17–40in exon 3. The sense primer for IGF-2, IF1 (58-CTGG-TGGACACCCTCCAGTTC-38), corresponded to se-quence 109–129 in exon 5, whereas that of IF2 (58-GCC-CACGGGGTATCTGGGGAA-38) corresponded to se-quence 322–333 in exon 6.

Expression of asparagine synthetase provided an in-ternal control for the amount of RNA template in eachpolymerase chain reaction (PCR) reaction. Primer A1(58-ACATTGAAGCACTCCGCGAC-38) correspondedto nucleotides 674–793 in exon 4. Anti-sense primer A3(58-AGAGTGGCAGCAACCAAGCT-38) correspondedto sequences 968–986 in exon 6. Anti-sense primer A4(58-CCTGAGGTTCTTCACAG-38) corresponded to se-quences 867–886 in exon 5. Asparagine primers A1 andA3 provided a control for the PCR for IGF-1 receptor andIGF-2. Primers A3 and A4 were used as a control forIGF-1. PCR was performed as described previously [25].All samples were analyzed at least in triplicate, and anarithmetic mean was used to calculate the representativevalues.

The kinetics of the various reactions were evaluatedusing the control cell line c-DNA to determine the opti-mal cycle number to which cDNA products should beco-amplified, while ensuring that production of the PCRproducts of interest (IGF-1 R, IGF-1, and IGF-2) as wellas the internal control AS remained exponential [25].PCR yields in the control cell lines were plotted on semi-logarithmic graphs to determine the appropriate cyclenumbers for evaluation of relative levels of expression inpatient samples. Each assay was carried out at three dif-

ferent cycle numbers from 22 to 28 cycles to ensure alinear range for the tumor analyzed. Since the quantity ofamplified AS product is assumed to be proportional tothe amount of initial mRNA template, a relative amountof IGF-R, IGF-1, and IGF-2 product could be determinedby normalizing IGF amounts to AS, as determined onlaser densitometry quantification of PCR products fol-lowing acrylamide gel electrophoresis. Finally, relativeamounts of IGF-R, IGF-1, and IGF-2 were normalized tothe levels found in cell line controls (MCF-7, RPMI7666, and NCI H69, respectively) (Figs. 1, 2).

Statistical Methods

Comparison of expression of IGF-R, IGF-1, and IGF-2in primary and metastatic tumors was performed usingthe Wilcoxon signed rank test. Correlation between IGF-R, IGF-1, and IGF-2 expression was evaluated using theSpearman correlation coefficients.

RESULTS

As shown in Table I, samples were available from 48patients. In 44 cases, only single time point specimenswere available for analysis. Thirty-six of these singlespecimens were derived from a primary tumor, and eightspecimens were obtained from metastases for which noprimary tumor was available.

In four cases, specimens were obtained from the pri-mary tumor and from the metastatic lesion. Overall, 52specimens were available from 48 patients. In total, 40samples were obtained from primary tumors, and 12specimens were taken from metastases.

Expression of IGF-1 R, IGF-1, and IGF-2 was de-tected in each tumor tested. As seen in Table I, relativeexpression for IGF-1 R varied over more than a 20-fold

Fig. 1. IGF-1 expression in osteosarcoma. Gel electrophoresis results for control RPMI 7666 cell lines and four tumor samples (364, 524, 495,and 317) analyzed for IGF-1. Cycle numbers for the polymerase chain reaction are evident at the top of the gel. Quantitation by laserdensitometry showed that tumors 364 and 524 expressed IGF-1 at lower levels of expression than the control, whereas 495 and 317 expressedthe ligand at higher levels.

IGF-1 and IGF-2 in Osteosarcoma 23

range, from a minimum of 0.2 to a high of 5.2 (with acomparative level of expression equaling 1 for the posi-tive cell line, MCF-7). IGF-1 relative expression variedover a similar range, from a minimum value of 0.2 to amaximum of 4.6 (relative to a value of 1 for RPMI 7666).IGF-2 variation was similar, from a minimum value of0.1 to a maximum value of 3.1 (relative to the control cellline NCI H69).

Comparison of IGF-R, IGF-1, and IGF-2 did not differsignificantly between primary and metastatic lung le-sions. The mean value for IGF-R expression in primarylesions was 1.1 and in metastases was 1.3 (P 4 0.74,Wilcoxon signed rank test). In primary tumors, IGF-1expression averaged 1.4, compared with 1.0 in metasta-ses (P 4 0.9). Comparable mean values for IGF-2 were0.7 in primary osteosarcoma and 0.7 in metastases (P 40.3).

In the four cases, (#s 3, 11, 14, and 15), for which bothprimary and metastatic lesions were available, no con-sistent changes in patterns of expression were found fromprimary to metastatic tumor (Table I). In case 3, IGF-Rwas estimated as 1.6 in the biopsy sample and 2.3 in themetastasis. IGF-1 was 2.5 in the primary and 2.0 in themetastasis. IGF-2 was not analyzed in the metastasis.

For case 11, both IGF-R and IGF-1 increased from theprimary to the metastasis (0.5 to 1.5 for IGF-R and 1.0 to2.0 for IGF-1), whereas IGF-2 decreased (1.5 to 0.3). Incase 14, IGF-R decreased from biopsy to metastasis (5.3to 2.7), whereas IGF-1 increased (1.5 to 3) and IGF-2decreased (1.6 to .0.5). Finally in case 15, IGF-R andIGF-2 remained consistent (0.9 to 1.0 for (IGF-R, and 1.6to 1.7 for IGF-2) in the primary and metastasis, whereasIGF-1 increased somewhat (1.8 to 3.3). The only notabletrend in the four tumors analyzed was increased expres-sion of IGF-1 in three tumors from the initial to themetastatic sample.

Evaluating the potential for autocrine and paracrinestimulation of proliferation in the samples analyzed, 21of 46 tumor specimens had IGF-R levels of expression

equal to or greater than MCF-7 cell lines. Twenty-sevenof 44 specimens had IGF-1 expression equal to or greaterthan the RPMI 7666 cell lines, and 21 of 38 samples hadlevels of IGF-2 expression equal to or greater than theNCI H69 cell line. There was a modest correlation be-tween the level of expression of IGF-R and IGF-1 (0.38,Spearman correlation coefficient), but no evident corre-lation between IGF-R and IGF-2 (0.06, Spearman corre-lation coefficient).

DISCUSSION

The outcome of osteosarcoma management has im-proved substantially with the advent of adjuvant and neo-adjuvant chemotherapy. A randomized trial of the effi-cacy of chemotherapy in the management of this sarcomademonstrated a definite survival advantage for patientsmanaged with adjuvant drug treatment that has persistedat long-term follow-up [32,33]. Despite the benefit ofchemotherapy, however, approximately 30–40% of pa-tients who present with localized disease will developlung metastases despite cytotoxic therapy [1]. Since thesepatients already receive multiagent chemotherapy atmaximally tolerated doses, it is unlikely that further im-provement in survival will be achieved by intensifyingcurrent drug treatment. In developing novel therapeuticapproaches, it is reasonable to determine whether bio-logical factors that stimulate osteosarcoma proliferationand metastasis can be identified and modified to achievea clinical advantage.

There is strong empirical evidence that the IGF signaltransduction pathway may be important in osteosarcoma.The highest incidence of osteosarcoma is found in thesecond decade of life at a time when adolescent skeletalgrowth acceleration is induced by increased productionof GH and elevated serum levels of IGF [34–36]. Themost common anatomic sites for development of osteo-sarcoma are those that respond to this increase in serumlevels of IGF with the maximal epiphyseal growth re-

Fig. 2. IGF-2 expression in osteosarcoma. Gel electrophoresis results for NCI H69 control line and three tumors (471, 74, and 35) analyzedfor IGF-2. Quantitation by laser densitometry showed low expression in tumors 34 and 35 relative to the control cell line and higher levels ofIGF-2 expression in 471.

24 Burrow et al.

sponse (i.e., the distal femur and the proximal tibia) [36].Other investigators have suggested that osteosarcoma pa-tients have higher serum levels of IGF than the generalpopulation [37]. Our group has been interested in deter-

mining whether alteration in IGF physiology can have aneffect on the biological behavior of osteosarcoma.

We have previously shown that osteosarcoma celllines demonstrate a proliferative response to IGF in vitro[14]. We have quantitated IGF-R in three murine sar-coma models and have shown that the tumors withIGF-R levels equal to or greater than MCF-7 demonstrateinhibited local tumor growth when implanted in micewho have undergone hypophysectomy to lower serumIGF levels [26,27]. We have shown in two different mu-rine sarcomas that hypophysectomy decreases the devel-opment of metastases in the host [26,27]. Furthermore,the effect of hypophysectomy in decreasing metastaticpotential was partially reversed in the RIF murine sar-coma model by treating the hypophysectomized animalswith human GH [27]. We have also duplicated the effectof hypophysectomy in the RIF model by treating animalswith medication that suppresses GH without resorting tohypophysectomy [28]. Similar results in decreasing sar-coma growth in vivo using pharmaceutical methods tolower serum IGF have been observed by Pinski et al. [38]using human osteosarcoma cell lines implanted in immu-nosuppressed mice.

These results suggest that modification of the IGF sys-tem in vivo may offer promise as a nontoxic adjuvanttherapy for osteosarcoma. In developing this potentialtherapy, it is important to determine the proportion ofhuman tumors that may be IGF responsive. We havepreviously tested 29 human sarcomas (including 10 pri-mary osteosarcomas) for IGF-1 R, IGF-1, and IGF-2 ex-pression using reverse transcriptase (RT)-PCR [25]. Inthe current experiment we have extended this initial se-ries by analysis of a further 52 osteosarcoma samplestaken from 48 patients and have questioned whether IGFreceptor or ligand is expressed at higher levels in meta-static lesions than in primary tumors. It would be rea-sonable to assume that metastasizing clones of cellsmight develop the phenotype associated with metastasisby increasing endocrine or autocrine responsiveness toIGF. However, as shown in the results reported above,we were unable to identify differences between primaryand metastatic tumors in levels of expression of eitherreceptor or ligand in these experiments.

To provide a reference level for the biological signifi-cance of the RT-PCR results, we have utilized cell linesthat have been tested for IGF responsiveness and IGFligand production in vitro and in vivo. MCF-7, for ex-ample, is known to proliferate in vitro in response tosmall quantities of IGF-1 [30]. Conditioned mediumfrom both RPMI 7666 and NCI H69 is known to causeMCF-7 proliferation, and IGF production from these celllines has been quantitated in vitro [31]. Finally, ourgroup has recently shown that MCF-7 cells, implanted inscid/lit mice deficient for GH and IGF, grow more slowlythan in control animals [29]. These in vivo results con-

TABLE I. IGF Receptor and Ligand Expression in Primary andMetastatic Osteosarcoma*

Case no. Site IGF-R IGF-1 IGF-2

1 Primary 0.80 0.18 0.582 Primary 0.78 0.22 —3 Primary 1.62 2.54 —

Metastatic 2.25 2.04 —4 Metastatic 0.22 0.40 0.425 Metastatic 0.92 1.17 1.066 Primary 1.66 1.07 1.02

Metastatic 1.02 2.12 —7 Primary 0.34 — —8 Primary 0.08 2.82 0.559 Primary — 3.11 1.54

10 Metastatic 1.27 2.38 1.1311 Primary 0.49 0.98 1.56

Metastatic 1.46 2.06 0.3112 Primary 1.40 — —13 Primary 1.10 1.08 1.0314 Primary 5.27 1.25 1.56

Metastatic 2.74 3.05 0.4615 Primary 0.86 1.75 1.62

Metastatis 0.97 3.30 1.8016 Metastatic 1.20 0.34 1.5217 Metastatic 1.08 0.83 —18 Primary 0.73 0.95 0.3319 Primary 1.42 0.75 0.5720 Metastatic 1.11 0.52 0.0921 Primary 1.47 4.61 1.0822 Metastatic 1.09 0.68 0.2423 Primary 1.27 2.28 —24 Primary 1.07 2.59 2.2225 Primary 0.49 0.81 1.1626 Primary 0.89 4.53 1.5827 Primary 0.38 — —28 Primary 0.88 1.41 0.5529 Primary 0.72 0.88 0.7230 Primary 0.23 1.8131 Primary 0.43 — —32 Primary 0.64 0.78 1.0433 Primary 0.65 0.68 1.2634 Primary 0.80 — —35 Primary — 4.20 —36 Primary 1.32 — —37 Primary 1.44 1.35 —38 Primary 0.54 1.28 0.7939 Primary 1.24 1.24 1.5340 Primary 0.76 0.80 2.4941 Primary 0.67 0.73 0.1942 Primary 0.74 3.97 1.5243 Metastatic 0.82 0.97 0.3344 Primary — 1.70 —45 Primary — 2.09 0.3646 Primary — — 0.7347 Primary — — 3.1048 Primary — — 0.88

*All numbers represent ratio of densitometry readings of specimen tocontrol cell lines (normalized as described in text).

IGF-1 and IGF-2 in Osteosarcoma 25

firm the in vitro data. It would therefore be reasonable toexpect that tumors with IGF-1 receptor mRNA tran-scripts equal to or greater in number than MCF-7 wouldbe IGF responsive and that tumors with IGF-1 or IGF-2levels of expression equal to or greater than the RPMI7666 and NCI H69 control cell lines might be capable ofautocrine stimulation.

In determining whether the results of IGF-R RT-PCRadequately reflect IGF-R protein quantitation, we previ-ously compared the results of RT-PCR with competitivebinding studies and Scatchard plot analysis in 25 humansarcomas (including six osteosarcomas). The amount ofmaterial necessary for membrane extraction of proteinand competitive binding makes this method impossiblefor most of our clinical osteosarcoma specimens. How-ever, in the clinical specimens tested, a moderate to highcorrelation between the two methods was observed [25].We are therefore satisfied that the methods used aboveare adequate (given the small amounts of tumor availablefor analysis) for quantitation of IGF-R and ligands.

The results described above suggest that approxi-mately 50% of osteosarcomas are responsive to IGF andthat more than 50% of osteosarcomas produce levels ofligand capable of inducing an autocrine stimulation oftumor proliferation. Furthermore, the correlation ofIGF-R and IGF-1 expression suggests that a substantialproportion of tumors may be self-sufficient in providingboth ligand and receptor. Despite the seeming impor-tance of the IGF signal transduction pathway in osteo-sarcoma, there was no suggestion that ligand or receptorare expressed at a higher level in metastases than inprimary tumors.

These results have significance for the design of clini-cal methods to alter osteosarcoma growth and metastasisthrough the IGF pathway. Other investigators have foundsimilar results in giant cell tumors of bone [39] and inEwing sarcoma [40]. At present there are several poten-tial methods for altering serum IGF, either surgically (byhypophysectomy) or pharmacologically. However, theefficacy of lowering serum IGF may be decreased if thetumor is capable of autocrine stimulation through theproduction of ligands. It is possible that lowering of thebackground level of IGF ligand in the interstitial fluid bylowering serum IGF will be effective in decreasing theeffectiveness of IGF produced by the tumor. It wouldcertainly be more effective, however, to provide directinhibition of the IGF-1 receptor in designing pharmaco-logical strategies for IGF inhibition.

The finding that IGF ligand and receptor is not ex-pressed at increased levels in metastatic lesions may sug-gest that the IGF tyrosine kinase signal transduction sys-tem is maximally stimulated by the levels of IGF activityseen in primary tumors and that there is no competitiveadvantage offered by further activation through this sys-tem. Indeed, the fact that 5 of the 12 lung metastases

analyzed expressed IGF-R at levels lower than MCF-7suggests that metastases can occur without maximal IGFstimulation and that another growth factor pathway isprobably responsible for proliferation in a proportion ofosteosarcomas.

These results confirm, however, that about 50% ofosteosarcomas may be appropriate for trials of IGF-modifying therapy. The development of new pharmaco-logical agents and the availability of transgenic micewith deficient serum IGF should be useful in determiningwhich osteosarcomas may be most appropriate for noveltherapies involving alteration of IGF physiology.

CONCLUSIONS

Approximately 50% of osteosarcoma tumors expressIGF-R at high levels, and about 50% of tumors expresshigh levels of IGF-1 or IGF-2 ligands.

REFERENCES

1. Bramwell VHC: The role of chemotherapy in osteogenic sarcoma.Crit Rev Oncol Hematol 1995;20:61–85.

2. Baserga R: The insulin-like growth factor I receptor: A key totumor growth? Cancer Res 1995;55:249–252.

3. LeRoith D, Baserga R, Helman L, et al.: Insulin-like growth fac-tors and cancer. Ann Intern Med 1995;122:54–59.

4. Macaulay VM: Insulin-like growth factors and cancer. Br J Can-cer 1992;65:311–320.

5. Westley BR: Insulin-like growth factors: The unrecognized onco-genes. Br J Cancer 1995;72:1065–1066.

6. Daughaday WH, Rotwein P: Insulin-like growth factors I and II.Peptide, messenger ribonucleic acid and gene structures, serum,and tissue concentrations. Endocr Rev 1989;10:68–91.

7. Froesch ER, Schmid C, Schwander J, et al.: Actions of insulin-likegrowth factors. Annu Rev Physiol 1985;47:443–467.

8. Gammeltoft S, Christiansen J, Nielsen FC, et al.: Insulin growthfactor II: Complexity of biosynthesis and receptor binding, mo-lecular biology and physiology of insulin and insulin-like growthfactors. Adv Exp Biol 1991;293:31–44.

9. Rechler MM, Nissley SP: The nature and regulation of the recep-tor for insulin-like growth factors. Annu Rev Physiol 1985;47:425–442.

10. El-Badry OM, Minniti C, Kohn EC, et al.: Insulin-like growthfactor II acts as an autocrine growth and motility in human rhab-domyosarcoma tumors. Cell Growth Differ 1990;1:325–331.

11. Hishinuma A, Yamanaka T, Kasal K, et al.: Growth regulation ofthe human papillary thyroid cancer cell line by protein tyrosinekinase and cAMP-dependent protein kinase. Endocr J 1994;41:399–407.

12. Hwang C, Fang K, Li L, et al.: Insulin-like growth factor-I is anautocrine regulator for the brain metastatic variant of a humannon-small cell lung cell line. Cancer Lett 1995;94:157–163.

13. Kleinman D, Karas M, Roberts CT, et al.: Modulation of insulin-like growth factor I (IGF-1) receptors and membrane-associatedIGF-binding proteins in endometrial cancer cells by estradiol. En-docrinology 1995;136:2531–2537.

14. Pollak MN, Polychranos C, Richard M: IGF1: A potent mitogenfor osteosarcoma. J Natl Cancer Inst 1990;82:219–224.

15. Tricoli JV, Rall LB, Karakousis CP, et al.: Enhanced levels of IGFmRNA in human colon carcinomas and liposarcomas. Cancer Res1986;46:6169–6173.

16. Mathews LS, Norstedt G, Palmiter RD: Regulation of insulin-likegrowth factor gene by growth hormone. Proc Natl Acad Sci USA1986;83:9343.

17. Sussenbach JS: The gene structure of the insulin-like growth fac-tor. Prog Growth Factor Res 1989;1:33.

26 Burrow et al.

18. Rotwein P: Two insulin like growth factor I messenger RNAsexpressed in liver. Proc Natl Acad Sci USA 1986;83:77.

19. Pietrzkowski Z, Sell C, Lammers R, et al.: Roles of insulin-likegrowth factor (IGF-1) and the IGF-1 receptor in epidermal growthfactor stimulated growth of 3T3 cells. Mol Cell Biol 1992;12:3883–3889.

20. DeAngelis T, Ferber A, Baserga R: Insulin-like growth factor Ireceptor is required for the mitogenic and transforming activitiesof the platelet-derived growth factor receptor. J Cell Physiol 1995;164:214–221.

21. Morrione A, DeAngelis T, Baserga R: Failure of the bovine pap-illomavirus to transform mouse embryo fibroblasts with a targeteddisruption of the insulin-like growth factor I receptor genes. JVirol 1995;69:5300–5303.

22. Valentis B, Porcu P, Quinn K, et al.: The role of the insulin-likegrowth factor I receptor in the transformation by simian virus 40T antigen. Oncogene 1994;9:825–831.

23. Burgaud J, Resnicoff M, Baserga R: Mutant IGF-1 receptors asdominant negatives for growth and transformation. Biochem Bio-phys Res Commun 1995;214:475–481.

24. Nuenschwander S, Roberts CT, LeRoith D: Growth inhibition ofMCF-7 breast cancer cells by stable expression of an insulin-likegrowth factor I receptor antisense ribonucleic acid. Endocrinology1995;136:4298–4303.

25. Sekyi-Otu A, Bell RS, Ohashi C, et al.: Insulin-like growth factorI (IGF-1) receptors, IGF-1, and IGF-2 are expressed in primaryhuman sarcomas. Cancer Res 1995;55:129–134.

26. Pollak M, Sem A, Richard M, et al.: Inhibition of metastaticbehavior of murine osteosarcoma by hypophysectomy. J NatlCancer Inst 1992;84:966–977.

27. Sekyi-Otu A, Bell RS, Andrulis I, et al.: Metastatic behavior of theRIF-1 murine fibrosarcoma: inhibited by hypophysectomy andpartially restored by growth hormone replacement. J Natl CancerInst 1994;86:628–632.

28. Pollak MN: Somatostatin lowers metastatic rate in RIF murinesarcoma. (submitted).

29. Yang X, Beamer WG, Huynh H, et al.: Reduced growth of humanbreast cancer xenografts in hosts homozygous for thelit mutation.Cancer Res 1996;56:1509–1511.

30. Cullen KJ, Yee D, Sly WS, et al.: Insulin-like growth factor re-ceptor expression and function in human breast cancer. CancerRes 1990;50:48–53.

31. Reeve JG, Brinkman A, Hughes S, et al.: Expression of IGF andIGF-BP in human lung cancer cell lines. J Natl Cancer Inst 1992;84:675–679.

32. Link MP, Goorin AM, Miser AW, et al.: The effect of adjuvantchemotherapy on relapse free survival in patients with osteosar-coma of the extremity. N Engl J Med 1986;314:1600–1606.

33. Link M, Goorin AM, Horowitz M, et al.: Adjuvant chemotherapyin osteosarcoma. Clin Orthop 1992;270:8–14.

34. Polednak AP: Human biology and epidemiology of childhoodbone cancers: a review. Hum Biol 1985;57:1–26.

35. Price CHG: Osteogenic sarcoma: An analysis of the age and sexincidence. Br J Cancer 1955;9:558–5745.

36. Price CHG: Primary bone forming tumors and their relationship toskeletal growth. J Bone Joint Surg 1958;408:574–593.

37. Vassilopoulou-Sellin R, Wallis C, Samaan N: Hormonal evalua-tion of patients with osteosarcoma. J Surg Oncol 1985;28:209–213.

38. Pinski J, Schally AV, Groot K, et al.: Inhibition of growth ofhuman osteosarcoma by antagonists of growth hormone-releasinghormone. J Natl Cancer Inst 1995;87:1787–1794.

39. Middleton J, Arnott N, Walsh S, Beresford J: The expression ofmRNA for insulin-like growth factors and their receptor in giantcell tumors of human bone. Clin Orthop 1996;322:224–231.

40. Scotlandi K, Benini S, Sarti M, et al.: Insulin-like growth factor Ireceptor mediated circuit in Ewing’s sarcoma/peripheral neuroec-todermal tumor: A possible therapeutic target. Cancer Res 1996;56:4570–4574.

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