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Published ahead of print on October 28, 2004, doi:10.1165/rcmb.2004-0232OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 32, pp. 65-71, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2004-0232OC

The Consequences of Insulin-Like Growth Factors/Receptors Dysfunction in Lung Cancer

Jasminka Pavelic, Simun Krizanac, Sanja Kapitanovic, Ljubomir Pavelic, Miroslav Samarzija, Fadila Pavicic, Sime Spaventi, Marko Jakopovic, Zlata Herceg-Ivanovi and Kresimir Pavelic

Division of Molecular Medicine, Rudjer Boskovic Institute; Clinical Hospital of Pulmonary Diseases Jordanovac; and Croatian Academy of Sciences and Arts, Division of Medical Sciences, Zagreb, Croatia

Correspondence and requests for reprints should be addressed to Professor Jasminka Pavelic, Division of Molecular Medicine, Rudjer Boskovic Institute, Bijenicka 54, P. Box 180, HR-10002 Zagreb, Croatia. E-mail: jpavelic{at}irb.hr


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The aim of this study was to investigate the consequences of insulin-like growth factors (IGF) and IGF receptor dysfunction in lung carcinomas. A correlation between increased expression (at mRNA and protein levels) for IGF-1 and IGF-1R and decreased apoptosis were found in large-cell carcinomas and adenocarcinomas. In 40% of informative adenocarcinomas expressing the highest values of IGF-2 and Ki-67 proteins, M6P/IGF-2R gene had LOH at one allele and a mutation in another allele. All four squamous cell carcinoma samples expressed LOH/mutation in the M6P/IGF-2R gene. The {alpha}IR3 strongly diminished proliferation and increased apoptosis in cultures established from squamous cell carcinomas overexpressing IGF-2 and IGF-1R. Telomerase activity was assessed in four squamous cell carcinomas. Cell treatment with IGF-1 increased telomerase activity. The opposite was observed when the cells were treated with {alpha}IR3, which inhibits the activity of IGF-1 receptors. Our findings suggest that disruption of the IGF/IGF receptors axis is involved in lung cancer formation.

Key Words: insulin-like growth factors • receptors • lung cancer


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Insulin-like growth factors (IGF) and their receptors and binding proteins play important roles in normal cellular and somatic growth. Consequently, their disruption could trigger mechanisms that lead to human carcinoma development. As potent mitogens, IGF (IGF-1 and IGF-2) exert their action through interaction with IGF receptor type 1, which has been identified as a potential control point for transformed cells and is a well defined target of IGF-2–modulated cell growth. IGF also bind with high affinity to binding proteins (IGF BP) that carry IGF in the blood circulation and regulate their availability to the specific receptors.

Perturbation in the level of any peptide from the IGF family seems to be implicated in lung cancer formation as endocrine, paracrine, or autocrine stimulators: IGF ligands and IGF-1 receptor through their mitogenic and anti-apoptotic action, and the M6P/IGF-2R as a tumor suppressor (1). The IGFBP, which enhance and inhibit the physiologic and biologic actions of IGF, have been shown to be secreted in vitro by a wide range of lung tumors (2).

Small-cell lung cancer (SCLC) cell lines secrete and respond to exogenous IGF, indicating an autocrine role for IGF-peptides in cancer cell proliferation. According to Reeve and colleagues (3), ~50% of SCLC and 30% of non-SCLC (NSCLC) cell lines show IGF-2 gene expression. Lung tumor cell lines also secrete IGF BP (3–5). These proteins bind the IGF and modulate the physiologic and cellular actions of these peptides. Lung cancer also demonstrates loss of imprinting at the IGF-2 locus (6).

An alteration of the M6P/IGF-2R gene is involved in lung cancer development. M6P/IGF-2R loss of heterozygosity (LOH), coupled with an intragenic loss-of-function mutation in the remaining allele, has been found in squamous cell carcinoma of the lung (7). An adenine-to guanine transition at exon 40 was found in one lung adenocarcinoma cell line resistant to growth inhibition by transforming growth factor (TGF)-ß (8). Finally, LOH at the M6P/IGF 2R locus predisposes patients to radiation-induced lung injury (9). Thus, loss of function of this receptor could significantly alter normal cell growth.

Taking into account that some lung cancer cells produce IGF-1R and IGF-2, which stimulate cell proliferation by an autocrine mechanism, and that cancer cell proliferation can be abrogated or alleviated by blocking the mRNA activity of these genes (1), the aim of our study was to investigate the consequences of function/dysfunction of the IGF's family of genes on the behavior of three types of lung cancers. We investigated whether (1) IGF 1/IGF 1R mRNA/protein expression influences the intensity of cell proliferation and apoptosis, (2) genetic changes in M6P/IGF-2R gene influence tumor cell proliferation, (3) telomerase activity in tumor cells could be modulated by changing the activity of IGF-1 or IGF-1R, and (4) disruption of IGF function could influence lung cancer cell growth.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Patients and Tissue Specimens
A total of 38 human lung carcinoma tissue specimens (15 adenocarcinomas, 19 large-cell carcinomas, and four squamous cell carcinomas) obtained from the Croatian Human Tumor Bank were analyzed. Tumor histopathology was confirmed by a pathologist (S.K.). The samples were obtained from patients treated surgically (adenocarcinoma: 11 men 42–72 yr of age [mean 59 yr] and four women 49–70 yr of age [mean 63 yr]; large-cell carcinoma: 12 men 43–70 yr of age [mean 56 yr] and seven women 44–78 yr of age [mean 61 yr]; squamous cell carcinoma: four men 45–79 yr of age [mean 71 yr]). The tissues were snap frozen in liquid nitrogen shortly after surgical removal, brought immediately to the laboratory, and stored at –80°C. Part of each frozen tumor sample was embedded in paraffin for immunohistochemical analysis. All other procedures were performed on frozen samples. For each tumor sample, normal tissue not immediately adjacent to cancer cells was used as a control for LOH/mutation analysis. Sections of each paraffin block were stained with hematoxylin and eosin to confirm malignant areas in the section. All persons gave their informed consent before inclusion in the study. A local ethics committee approved the study.

RNA Extraction and Reverse Transcription Polymerase Chain Reaction
For RNA extraction, tissue samples were homogenized in 1 ml of solution containing 4 M guanidinium thiocyanate, 25 mM sodium citrate (pH 7.0), 5 g/l sarkosyl, and 100 mM 2-mercaptoethanol (10). Reverse transcription (42°C for 1 h) for all IGF RNAs was performed as described previously (1, 11, 12). The cDNA fragments were of the following sizes: IGF-1, 397 bp; IGF-1R, 729 bp; ß-actin, 317 bp.

Immunohistochemistry
Immunohistochemical tests were performed on formalin-fixed, paraffin-embedded tissue using the avidin-biotin-peroxidase method. Sections, cut at 4 µm, were subjected to a heat-induced epitope retrieval technique in 10 mM citrate buffer (pH 6.0) in an 850 W microwave oven for 10 min. Anti–IGF-1 (goat; 50 µg/ml) (R&D Systems, Minneapolis, MN), anti–IGF-2 (mouse) (Upstate Biotechnologies, Lake Placid, NY), and anti-Ki-67 (mouse, 10 µg/ml) (DAKO Cytometrics, Glostrup, Denmark) monoclonal antibodies were diluted 1:50 and incubated for 15–18 h. Anti–IGF-1R (Santa Cruz Biotechnologies, Santa Cruz, CA) were diluted 1:100 and incubated for 15–18 h. Detection was achieved using the DAKO LSAB 2 kit (Carpinteria, CA). Anti–M6P/IGF 2R rabbit polyclonal antibody was a generous gift from Zeneco (Houston, TX; www.geneimprint.com). Detection was achieved using the DAKO LSAB 2 kit according to the manufacturer's instructions. Negative controls were stained by substitution of the primary antibodies with nonimmune mouse or rabbit immunoglobulins. Appropriate positive controls (hemangiosarcoma tissue for IGF-1, Wilms' tumor for IGF-2, thyroid gland tissue for IGF-1R, normal colon for M6P/IGF-2R, and lymph nodes or skin for Ki-67) were stained positively. The tumor cells showed strong diffuse cytoplasmic immunopositivity for IGF-1, IGF-2, M6P/IGF-2R, and Ki-67, and cytoplasmic and focal membranous reactivity for IGF-1R. The intensity of staining was subjectively judged as: negative (0), weak (1), moderate (2), and strong (3).

Loss of Heterozygosity Analysis and Mutation Detection in M6P/IGF-2R
We used two polymorphisms present close together in the 3'-untranslated region of the human M6P/IGF-2R receptor gene to determine, by polymerase chain reaction (PCR) amplification of DNA, the frequency of LOH. One polymorphic site is a dinucleotide (GT) repeat sequence, and the other is a tetranucleotide insertion/deletion (ACAA) site 5' of the GT repeat. Together these polymorphisms give observed heterozygosity of ~ 65%. The forward and reverse PCR primers used were (5'-TTGCCGGCTGGTGAATTCAA-3') and (5'-CTCTTCAGGTTCTCATGATA-3'), respectively. The reaction conditions for PCR and a detailed method description were published by Kong and colleagues (7). Allelic loss was defined as a > 50% decrease in the ratio of the two alleles in the tumor versus its corresponding normal tissue.

In tumors with LOH at one M6P/IGF-2R locus, the remaining allele was screened for mutations in the ligand binding regions by direct sequencing of PCR products according to Kong and colleagues (7). The regions screened for mutations were exons 8–11, exons 27–29, exon 31, exons 33 and 34, and exons 37–39. The exon-specific forward and reverse PCR primers used have been published (13).

Cultivation of Lung Cancer Cells: Cell Proliferation, Apoptosis, and Telomerase Activity After Blocking of IGF-1R by Monoclonal Antibodies
The procedure for tumor cell cultivation has been described (1). Cells were kept in T-75 flasks with RPMI 1640 medium supplemented with 10% fetal bovine serum and 5% human serum, 1% glutamine, and 20 mM HEPES. Tumor cells were maintained as monolayers. For the purpose of specific analysis, tumor cells were seeded in 96-well plates (2 x 103 cells/well for cell proliferation assay-MTT test; 2 x 105 cells/well for apoptosis and telomerase assay) and incubated for 4 d in RPMI medium (containing 0.1% bovine serum albumin, 5 µg/ml transferrin, 5 ng/ml selenic acid, and antibiotics) with or without addition of 1,000 ng/ml of {alpha}IR3 monoclonal antibodies (AMS Biotechnology, Lugano, Switzerland). At the end of incubation, MTT test and telomerase or apoptosis assay were performed.

To determine the influence of IGF-1 on telomerase activity, 5 x 105 cells were plated (in 1 ml of medium) per well using 24-well plates, and cells were grown to 70% confluence and placed in serum-free medium for 10 h before treatment with 10, 100, and 1,000 ng/ml of IGF-1 for 24 h. The experiment was done in triplicate twice.

MTT Assay
Cell number, reflecting cell proliferation capability, was determined by the MTT dye reduction assay. The absorbance was measured at 570 nm with an ELISA plate reader (Multiscan MS; Labsystems, Franklin, MA). Data were expressed as the percentage of decrease in cell growth compared with untreated control cells.

Analysis of Apoptosis
FACScan analysis of apoptosis was performed according to the procedure of Sard and colleagues (14). Two million cells per sample were fixed with 2% paraformaldehyde in phosphate-buffered saline, washed twice with Tris-buffered saline (50 mM Tris HCl in saline solution), and permeabilized for 1 min with ice-cold acetone. Staining was performed by incubating cells for 1 h at 37°C in 25 µl of TUNEL reaction mixture (in situ Cell Death Detection Kit; Boehringer, Mannheim, Germany). Samples were analyzed by FACScan (Becton Dickinson, Erembodegem-Aalst, Belgium).

Telomerase Assay
The telomerase assay was performed with a commercial telomerase PCR ELISA kit (Roche Molecular Biochemicals, Indianapolis, IN) according to Bosserhoff and colleagues (15) and the manufacturer's protocol. Absorbance at 450 nm was measured using an ELISA plate reader (Multiscan MS) (16). Cells were lysed, and protein concentrations were determined by Bradford assay. Two micrograms of protein were used for each telomerase PCR. Telomerase assay was performed using positive and negative controls as specified in the kit.

Statistical Analysis
The data are shown as mean ± SD. Student's t test (in SAS/Stat) was performed to compare the results between two groups of tumor samples. The level of significance was set at P < 0.01. Box-Whisker plots were generated in the basic module of the program Statistica. Correlation between apoptosis and IGF's gene status was analyzed with the Wilcoxon rank-sum test. Correlation between telomerase activity and IGF-1 was calculated with the Pearson correlation coefficient.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Large-Cell Carcinoma and Adenocarcinoma
The relation among IGF-1/IGF-1R protein and mRNA expression and apoptosis. The correlation between increased expression of protein for IGF-1 (P = 0.09869) and IGF-1R (P = 0.02731), and a decreased number of apoptotic cells was found in adenocarcinoma samples of different stages (Figure 1, Table 1). Similar results were obtained at the level of mRNA (Table 1). Statistically significant positive correlations were found between apoptosis and IGF-1 mRNA expression (P = 0.03578) and for IGF-1R mRNA expression (Figure 2) (P = 0.02731). The level of receptors and IGF-1, measured through protein and mRNA expression, were of the same intensity in the majority of samples. There was no correlation between tumor stage and protein/mRNA expression or apoptosis (Table 1).



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  		Figure 1. The relation between IGF-1 (A) and IGF-1R (B) protein expression (immunohistochemical analysis) and apoptosis frequency in adenocarcinoma.

 

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  		TABLE 1. IGF-1, IGF-1r, and apoptosis in different stages of lung adenocarcinoma{ddagger}

 


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  		Figure 2. The relation between IGF-1 (A) and IGF-1R (B) mRNA expression and apoptosis frequency in adenocarcinoma (1: samples negative for IGF-1/IGF-1R mRNA; 2: samples positive for IGF-1/IGF-1R mRNA).

 
Similar results were also observed in the group of 19 large-cell lung carcinomas. Higher IGF-1 (P = 0.00887) and IGF-1R (Figure 3) (P = 0.0272) expression diminished cell apoptosis and correlated with proliferation index measured through Ki-67 protein expression (Table 2).



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  		Figure 3. The relation between IGF-1 (A) and IGF-1R (B) (immunohistochemical analysis) and apoptosis in large-cell carcinoma.

 

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  		TABLE 2. Apoptosis rate and IGF-1/IGF-1r/KI-67 protein status in patients with large-cell carcinoma

 
M6P/IGF-2R loss of heterozygosity/mutations analysis. Fifteen adenocarcinomas and corresponding normal tissue samples were analyzed. Ten normal tissues were informative (heterozygous) for two polymorphic markers in M6P/IGF-2R gene, and four of them (40%) had LOH at the M6P/IGF-2R locus in the tumor samples (Table 3). The remaining allele in 4 out of 10 LOH samples was screened for mutations in the ligand-binding region of the M6P/IGF-2R gene. Two alleles had GCC to ACC mutation in exon 34, which results in Ala-1618 Thr substitution. One allele had GGT to CGT mutation in exon 27, which results in Gly-1296 Arg substitution, and one had G insertion in the poly-G repeat region (nucleotides 4,089–4,096) of exon 28 (Table 3). The remaining alleles of the tumor tissues whose corresponding normal samples were noninformative for two polymorphic markers tested (five samples) were of the wild type, the same as the six informative tumor samples without LOH.


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  		TABLE 3. IGF-2 protein expression and status of M6p/IGF-2r gene mutations in lung adenocarcinomas

 
LOH/mutations did not correlate with tumor stage. However, the amount of IGF-2 protein and the proliferation index (Ki-67) were of highest values in tumors with LOH in one and point mutation in the other allele. Contrary to this, tumors with neither LOH nor mutation expressed lower values of IGF-2 protein and proliferation index.

Squamous Cell Carcinoma
The relation among IGF-2/IGF-1R protein expression, apoptosis, and mutations in the M6P/IGF-2R gene. Immunohistochemical reactions to IGF-2 and IGF-1R were of the highest values in all four squamous cell carcinoma samples tested. Loss of heterozygosity at one allele and mutation in the other M6P/IGF-2R allele were found in all four samples (Table 4).


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  		TABLE 4. IGF-2 and IGF-1r protein expression, M6p/IGF-2r gene status, and response to IGF-1r monoclonal antibody treatment of primary cell culture derived from patients suffering from different tnm stage of squamous cell carcinoma

 
Effect of the IGF-1 receptor-blocking antibody on cell proliferation and apoptosis. Tumor cell cultures were established from all four squamous cell carcinomas. To investigate whether blockade of the IGF-1 receptor has an affect on cell proliferation and apoptosis, a monoclonal antibody ({alpha}IR3) that blocks the binding sites of the IGF-1 receptor was used. Cell proliferation was strongly diminished (by 70–95%), whereas apoptosis increased (44–70%) after treatment with monoclonal antibodies (Table 4).

Modulation of telomerase activity by IGF-1/IGF-1R. Telomerase activity was assayed in all four squamous cell carcinoma primary cultures. The influence of cell treatment with IGF-1 on telomerase activity is shown in Figure 4. Relative telomerase activity was IGF-1–dose dependent; it increased from 100% to 300%, with the increase of the amount of IGF-1 added to the cell culture established from a sample obtained from patient number 1, as indicated in Table 4 (R2 = 0.9939). The highest dose of IGF-1 (1,000 ng/ml) applied to the other three primary cultures established from squamous cell carcinoma samples increased the relative telomerase activity to 240, 190, and 150% (data not shown). The opposite was observed when the cells were treated with {alpha}IR3 monoclonal antibodies that inhibit the activity of IGF-1 receptors. Before treatment with {alpha}IR3, the median telomerase activity was 280. After treatment, the value representing relative telomerase activity decreased to 100. The results were statistically significant (Figure 5) (P = 0.03122).



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  		Figure 4. Modulation of telomerase activity by IGF-1 in squamous cell carcinoma. Telomerase activity was assayed in the response to 10, 100, and 1,000 ng/ml IGF-1 treatment for 24 h. The experiment was done in triplicate.

 


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  		Figure 5. Inhibition of telomerase activity in squamous cell carcinoma by {alpha}IR3 monoclonal antibody. The values represent the mean of data obtained in experiments done with primary cultures established from four different squamous cell carcinoma samples.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
IGF-1 secreted by host or tumor cells plays a major role in tumor progression. Most, if not all, of the effects of IGF-1 result from its activation of the IGF-1R, whose signaling is required for maintenance of growth and tumorigenicity of many tumors, including lung cancer. There are numerous reports demonstrating this connection for a variety of human tumors (1, 11, 12, 17, 18). For example, Miyamoto and coworkers (19) have established a correlation between high levels of circulating IGF-1 and low levels of IGFBP3 and risk of developing lung cancer, which are known to produce matrix metalloproteinase-7. In turn, in lung cancer cells overexpressing IGF-1R, inhibition of phosphorylation of IGF-1R, and inactivation of the downstream molecule Akt could be achieved by inhibition of IGF-1 binding to the IGF-1R. Similar are the data showing that IGF-1 and IGF-2 stimulate expression and activity of metalloproteases, pointing that IGF-1 may promote lung cancer invasiveness. Results from other studies in lung cancer cell lines (20) and human population studies (21) support the role of IGF in lung cancer development.

Triggering apoptosis in response to aberrant expression of oncogenes is an essential part of tumor suppression. From this, it is obvious that another mechanism by which IGF-1/IGF-1R could promote tumorigenicity is inhibition of apoptosis, as it was shown for NSCLC (22). Similarly, inhibitors of IGF-1R and c-kit applied to SCLC cells caused proliferation inhibition and apoptosis induction (23), as did transactivation of IGF-1R by amphiregulin in NSCLC (22). In addition, Min and coworkers (24) have shown successful therapy in a lung cancer xenograft model using recombinant adenovirus expressing truncated dominant negative IGF-1R. Such receptors suppressed tumorigenicity in vivo and in vitro, upregulated apoptosis, and blocked IGF-1 and IGF-2 induction of Akt-1. In addition, epidemiologic data suggest that high levels of IGF-1 constitute a risk factor for lung cancer and other cancer development (25).

That a functional IGF-1R is required for successful cell transformation is illustrated in an experiment where IGF IR–/– cells did not undergo transformation even when overexpressing SV 40 T antigen, activated ras, or epidermal growth factor or platelet-derived growth factor receptors. In this case, transformation seems to involve autocrine stimulation because IGF-1 production and secretion is increased in normal cells after transformation by SV40 T antigen, but the null cells cannot respond to the IGF-1 and do not transform. Additionally, embryonic fibroblasts derived from IGF-1R knockout embryos (R-cells) do not grow in serum-free medium, despite supplementation with growth factors, and grow more slowly in media containing serum than wild-type cells (26).

The results of our study indicate the importance of IGF-1/IGF-2/IGF-1R molecules in the pathogenesis of lung cancer. Understanding the role and mechanism of action of IGF in the development and progression of lung cancer is slowly advancing. IGF-1 and IGF-2 autocrine stimulation through IGF-1R, found in some human tumors of epithelial origin (27, 28), is plausible also for lung cancer (1). Recently, we showed a similar pattern of IGF action in hemangiopericytomas and in gastric cancer (11, 12, 17). In this article, we do not show directly the existence of an autocrine loop of IGF action. Rather, we show the intracellular overproduction of IGF-1, IGF-2, and IGF-1R. However, because two indicators of transformed cell phenotype were recorded (increased proliferation and decreased apoptosis), it is reasonable to believe that IGF-1/IGF-2/IGF-1R do have a role in the pathogenesis of lung cancer. However, the molecular mechanism by which IGF-1/IGF-2/IGF-1R is increased in these tumors is unknown. Only amplification of the IGF-1R locus has been reported in a small number of breast cancers and melanoma cases (29).

Contrary to IGF-1R, M6P/IGF-2R mediate activation of the growth inhibitor TGF-ß and clearance of IGF-2 (30). Therefore, it is reasonable to believe that dysfunction of M6P/IGF-2R could contribute to tumor development. M6P/IGF-2R has been shown to be mutated in a number of human tumors, including squamous cell carcinoma of the lung (11). Our results are in agreement with these data. We confirmed that bioavailability of IGF is critical in tumor development by the observation of mutated forms of IGF-2R in tumors in which IGF-2 and/or IGF-1R were overexpressed. The mutations were found in both alleles of this tumor suppressor gene, consistent with Knudson's two-hit hypothesis model. The loss of anti-oncogenic activity and, as a consequence, neoplastic transformation in lung has been shown by other authors (7, 31).

Telomeres are crucial to the life of the cell because they shorten throughout a cell's mitoses and impose a finite life span of normal cells. In turn, cancer cells are able to maintain the length of their telomeres with the aid of the enzyme telomerase. Therefore, telomerase activity is closely linked to attainment of cellular immortality, a step in cancerogenesis. Acting in concert with oncogenes or mutated tumor suppressor genes, telomerase expression is associated with the malignant phenotype and is found in up to 90% of all human cancers where it is 50–80% more active than in normal cells. The increased telomerase activity found in four squamous cell carcinoma primary cell lines tested confirms the current concept that this enzyme is necessary for maintenance of the malignant phenotype.

Due to the concept that telomerase activation leads to cell immortalization, interference with telomerase activity could represent a target for cancer therapy. There are numerous articles describing such approaches. However, we have taken an alternative approach. Because we have shown that telomerase activity is increased by augmenting the amount of IGF-1 operating through IGF-1R on the cancer cells, we attempted to eliminate telomerase activity by blocking IGF-1R, which was shown to be overexpressed in squamous cell carcinoma of the lung. Monoclonal antibody to IGF-1R diminished telomerase activity, indicating that it abrogated IGF-1/IGF-1R cancer-protective effect. It should be therefore proposed that the mechanism of squamous cell carcinoma growth and survival is IGF-1/IGF-2/IGF-1R dependent and that one of the mechanisms by which IGF-1R exerts its action after ligand binding is mediated through telomerase activity. However, the intermediate molecules, processing the signals from IGF-1R to telomerase gene have to be further elucidated. They probably involve phosphatidylinositol 3'-kinase/Akt/nuclear factor {kappa}B signaling, as it was shown for SCLC cells (32) and multiple myeloma cell lines (33).

In conclusion, our results have shown that lung cancer cells overexpress IGF-1/IGF-2/IGF-1R, supporting their importance in cancer development and maintenance. In addition, simultaneous M6P/IGF-2R gene mutations may contribute to this process, providing further support for its function as a tumor suppressor. One of the mechanisms by which IGF-1/IGF-2/IGF-1R acts as a cancer-protector is activation of telomerase activity.


   Acknowledgments
 
The authors thank Dr. B.W. Ragland for correcting the grammar and syntax.


   Footnotes
 
This study was supported by grants 0098089, 0098092, 0098093, and 0101023 from the Ministry of Science and Technology, Republic of Croatia.

Conflict of Interest Statement: J.P. has no declared conflicts of interest; S.K. has no declared conflicts of interest; S.K. has no declared conflicts of interest; L.P. has no declared conflicts of interest; M.S. has no declared conflicts of interest; F.P. has no declared conflicts of interest; S.S. has no declared conflicts of interest; M.J. has no declared conflicts of interest; Z.H-I. has no declared conflicts of interest; and K.P. has no declared conflicts of interest.

Received in original form July 22, 2004

Received in final form October 20, 2004


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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