<p>Genetic and epigenetic variation is a key determinant of human diversity and has an impact on disease predisposition. Single nucleotide polymorphisms (SNPs) and copy number polymorphisms (CNPs) are the main forms of genetic variation. The challenge is to distinguish normal variations from disease-associated changes. Combination of genetic and epigenetic alterations, often together with an environmental component, can cause cancer. In paper I, we investigated possible alterations affecting the transcriptional regulation of PDGFRα in patients affected with central nervous system tumors by characterizing the haplotype combinations in the <i>PDGFRA</i> gene promoter. A specific over-representation of one haplotype (H2δ) in primitive neuroectodermal tumors and ependymomas was observed, suggesting a functional role for the ZNF148/PDGFRα pathway in the tumor pathogenesis. In paper II, 50 glioblastomas were analyzed for DNA copy number variation with a chromosome 22 tiling genomic array. While 20% of tumors displayed monosomy 22, copy number variations affecting a portion of chromosome 22 were found in 14% of cases. This implies the presence of genes involved in glioblastoma development on 22q. Paper III described the analysis of copy number variation of 37 ependymomas using the same array. We detected monosomy in 51.5% of the samples. In addition, we identified two overlapping germline deletions of 2.2 Mb and 320 kb (the latter designated as Ep CNP). In order to investigate whether Ep CNP was a common polymorphism in the normal population or had an association with ependymoma development, we constructed a high-resolution PCR product-based microarray covering this locus (paper IV). For this purpose, we developed a program called <i>Sequence Allocator</i>, which automates the process of array design. This approach allowed assessment of copy number variation within regions of segmental duplications. Our results revealed that gains or deletions were identical in size and encompassed 290 kb. Therefore, papers I-IV suggest that some SNPs and CNPs can be regarded as tumor-associated polymorphisms. Finally, paper V describes variation of DNA methylation among fully differentiated tissues by using an array covering ~9% of the human genome. Major changes in the overall methylation were also found in colorectal cancer cell lines lacking one or two DNA methyltransferases.</p>

Astrocytic tumors are the most common malignancies of the central nervous system, accounting for more than 60% of all primary brain tumors. The prognosis for high grade astrocytoma patients is dismal and there is no curative treatment, today. A molecular hallmark of astrocytic tumors is dysregulated receptor tyrosine kinase signaling, especially of the epidermal growth factor receptor (EGFR, ErbB1). The aim of the present thesis was to identify endogenous human proteins that downregulate the function of the ErbB1 receptor. We identified a human gene family, the leucine-rich repeats and immunoglobulin-like domains family (LRIG), consisting of LRIG1, LRIG2 and LRIG3, which might fulfill this criterion. Two candidates were identified, LRIG1 and LRIG2, which genes were localized to regions frequently deleted in human cancers, chromosome bands 3p14 and 1p13, respectively. LRIG1 and LRIG2 mRNA were expressed in all tissues analyzed, with high expression in brain and other organs. The LRIG mRNA were predicted to encode integral membrane proteins. Antibodies against LRIG1 and LRIG2 were developed and the protein expression was analyzed. LRIG1 was found to have an apparent molecular weight of 143 kDa and was expressed in most tissues with high expression in glandular tissues of the breast and prostate, and the peptic cells of the stomach. LRIG2 was slightly smaller and had an apparent molecular weight of 132 kDa. The LRIG proteins were localized to the cell surface and to perinuclear structures, where LRIG1 co-localized with the trans-Golgi network and early endosomes. LRIG1 was found to restrict growth factor signaling by enhancing receptor ubiquitylation and degradation. We showed that LRIG1 interacted with all four members of the ErbB family and induced their downregulation. The interaction with ErbB1 involved both the LRR-domains and the Ig-like domains of LRIG1. LRIG1 enhanced receptor degradation by recruiting the E3 ubiquitin ligase c-Cbl to the LRIG1-ErbB1 complex. LRIG1, LRIG2, and LRIG3 were expressed in glioma cell lines and tumor tissues. The LRIG expression was analyzed in 404 astrocytic tumor samples. We found that perinuclear LRIG protein expression correlated with increased survival of patients with astrocytic tumors. Especially perinuclear LRIG3 showed strong correlations with patient survival and tumor cell proliferation index. In summary, this thesis contains the discovery and characterization of human LRIG1 and LRIG2. LRIG1 was found to interact with ErbB receptors and downregulate their function. In a clinical material, expression of LRIG proteins correlated with survival in patients with astrocytic tumors.

Background: Glioblastoma is the most common and aggressive type of glioma and associated with poor prognosis. Apart from ionizing radiation and some rare genetic disorders, few aetiological factors have been identified for primary brain tumours. Inverse associations to asthma and low IgG levels for varicella zoster virus have in previous studies indicated that the immune system may play a role in glioma development. Little is known about prognostic factors in glioma. Previous studies have shown an association between age, Karnofsky performance status, O6-methylguanine-DNA methyltransferase (MGMT) hypermethylation, and prognosis. Polymorphisms in different low penetrance genes have in some studies been associated with glioma prognosis. Material and methods: In paper I, we analysed IgG levels for four different viruses, Epstein-Barr virus (EBV), cytomegalovirus (CMV), varicella zoster virus (VZV) and adenovirus (Ad), in prediagnostic blood samples from 197 cases with glioma and 394 controls collected from three large cohorts: the Northern Sweden Health and Disease Study; the Malmö Diet and Cancer Study; and the Diet, Cancer and Health cohort from Copenhagen. ELISA was used to measure IgG levels and for EBV response to both the nuclear antigen (EBNA1) and the viral capsid antigen (VCA) was measured, for VCA using immunoflourescence. IgG levels were divided into quartiles and binary logistic regression was used to compare the quartiles in cases and controls. All odds ratios were adjusted for age, sex, and cohort. In paper II-IV, we studied 176 glioblastoma cases from Sweden and Denmark. We collected treatment and follow-up data on the cases. We genotyped 30 tagging SNPs in EGF, 89 in EGFR, 27 in VEGFR2, and 17 in VEGF. We also studied 1458 SNPs in 136 DNA repair genes. Hazard ratios were calculated using Cox regression; the major allele was set as categorical variable and all HR were adjusted for age, sex, country, and treatment. For the DNA repair gene results, we adjusted the p-values for multiple testing. Significant findings were confirmed in separate datasets. Results and Discussion: We found a trend towards higher IgG VZV levels in controls compared to glioma cases, especially when restricting the analyses to only include glioma cases with at least 2 years between blood sample and diagnosis. This finding might indicate that there is an aetiological and not a disease-related association. This confirms previous findings and support that a strong immune system can detect and inhibit growth of small cancer clusters. In EGF, we found seven SNPs in one haplotype block that were significantly associated with glioblastoma survival. Four of the SNPs were available for confirmation; however, none reached statistical significance. One explanation could be age differences in the different cohorts. In EGFR, four SNPs associated with survival were found; however, as 89 polymorphisms were tested this was the expected outcome by chance. In VEGF and VEGFR2, we found two SNPs associated with glioblastoma survival, but they could not be confirmed in the separate dataset, and due to multiple testing, were considered to be false positives. Among the DNA repair genes, we found nine SNPs in three genes-MSH2, RAD51L1 and RECQL4-associated with glioblastoma survival after confirmation and adjustment for age, sex, country, and treatment. After adjusting for multiple testing, one SNP in MSH2 and one in RECQL4 remained significant. Conclusions: Our studies provide additional knowledge to the aetiological and prognostic factors important for glioma, emphasising the possible importance of immune function mechanisms. We found limited evidence for the role of genetic variants in glioma progression genes, and some for DNA repair variants as prognostic factors for glioblastoma survival.

Genetic and epigenetic variation is a key determinant of human diversity and has an impact on disease predisposition. Single nucleotide polymorphisms (SNPs) and copy number polymorphisms (CNPs) are the main forms of genetic variation. The challenge is to distinguish normal variations from disease-associated changes. Combination of genetic and epigenetic alterations, often together with an environmental component, can cause cancer. In paper I, we investigated possible alterations affecting the transcriptional regulation of PDGFRα in patients affected with central nervous system tumors by characterizing the haplotype combinations in the PDGFRA gene promoter. A specific over-representation of one haplotype (H2δ) in primitive neuroectodermal tumors and ependymomas was observed, suggesting a functional role for the ZNF148/PDGFRα pathway in the tumor pathogenesis. In paper II, 50 glioblastomas were analyzed for DNA copy number variation with a chromosome 22 tiling genomic array. While 20% of tumors displayed monosomy 22, copy number variations affecting a portion of chromosome 22 were found in 14% of cases. This implies the presence of genes involved in glioblastoma development on 22q. Paper III described the analysis of copy number variation of 37 ependymomas using the same array. We detected monosomy in 51.5% of the samples. In addition, we identified two overlapping germline deletions of 2.2 Mb and 320 kb (the latter designated as Ep CNP). In order to investigate whether Ep CNP was a common polymorphism in the normal population or had an association with ependymoma development, we constructed a high-resolution PCR product-based microarray covering this locus (paper IV). For this purpose, we developed a program called Sequence Allocator, which automates the process of array design. This approach allowed assessment of copy number variation within regions of segmental duplications. Our results revealed that gains or deletions were identical in size and encompassed 290 kb. Therefore, papers I-IV suggest that some SNPs and CNPs can be regarded as tumor-associated polymorphisms. Finally, paper V describes variation of DNA methylation among fully differentiated tissues by using an array covering ~9% of the human genome. Major changes in the overall methylation were also found in colorectal cancer cell lines lacking one or two DNA methyltransferases.

Gene therapy has received much attention the last decade as a method to correct a number of disorders arising from a defective gene. Gene therapy can be defined as the introduction of a functional genetic element into a cell for a therapeutic purpose. This is a very broad term and gene therapy can be applied to a wide range of diseases from genetic diseases such as cystic fibrosis to infectious diseases or even acquired genetic diseases such as cancers. Adenoviruses (Ad) are the second most common vector for gene therapy in clinical trials today, and these vectors are mostly based on serotype 2 or 5 (Ad2 and Ad5). It has been shown that Ad2 and Ad5 use a receptor that is often downregulated in malignant cells and they also suffer from shortcomings because of the high levels of pre-existing immunity against these serotypes in the society. Hence, new and improved vectors serving as alternatives to these serotypes need to be developed to make gene therapy a successful treatment option. The work presented herein is devoted to analyse what alternative adenovirus vectors could be used for treatment of glioma brain tumours. A number of different adenovirus serotypes were screened for their ability to infect human glioma tumour cells in vitro. Established cell lines as well as low-passage glioma cells from different donors were used. Adenovirus serotype 11p (Ad11p) proved to be a promising vector candidate because of its capacity to efficiently infect the glioma cells and its low prevalence in the society. The complete genome of this serotype was sequenced to further develop this as an alternative adenovirus vector. Furthermore, a number of cell lines were produced to generate E1 deleted Ad11p vectors. Other promising vector candidates were Ad16 and a chimpanzee adenovirus called CV23. Ad16 was the most efficient human serotype to infect human low-passage glioma cells and the prevalence for this serotype is also very low. The overall most efficient virus was surprisingly the non-human CV23 virus. This adenovirus has no prevalence in humans, but efficiently infects human cells in vitro. The first analysis was made on established glioma cell lines and was followed up by using low passage glioma cells from a number of different patients. The glioma cells were analysed when subjected to &lt;20 passages (low passage) and then again at &gt;40 passages (high passage). The cells at a higher passage number were significantly more permissive to Ad5 than the cells analysed at a low passage number. This could in part explain why some of the promising in vitro data for Ad5 have shown a limited success in vivo. In contrast, CV23 infected the low and high passage gliomas equally. This indicates that CV23 uses an internalisation mechanism subjected to less variation than the mechanism used by Ad5. We further characterised the receptor interaction of CV23 and found that none of the previously known primary receptors for adenoviruses were of importance for binding. We found that bovine serum albumin present in the growth medium was responsible for the high binding capacity to cells. Binding is a criterion for the first step of the infection, but not necessarily a good correlate to the infection capacity. CV23 infected human cells efficiently also in the absence of bovine serum albumin.

Glioblastoma multiforme (GBM) is the most aggressive and most common primary brain tumor in adults accounting for 50-60% of primary brain tumors. The prognosis for patients with GBM remains poor and treatment is mainly palliative with a mean survival time of less than one year. Radiotherapy is used extensively in the management of glioblastoma either alone or in combination with surgery and/or chemotherapy. However, this tumor is one of the most resistant tumors to radiotherapy thus limiting the benefit of this form of treatment. <p>Studies have shown that malignant tumors have a high content of glutathione an antioxidant responsible for protecting the cells against damage from free radicals (mainly superoxide, hydroxyl and hydrogen peroxide). It is well established that glutathione, by neutralizing these free radicals plays a major role in radioresistance. Glioblastoma has relatively high levels of glutathione. In this study, by reducing the glutathione content of glioblastoma in a rat model, we were able to investigate the effect of this reduction in enhancing the effect of radiotherapy as a form of treatment for glioblastoma multiforme in a rat model. <p>By injecting L-Buthionine-SR-Sulfoximine (BSO) in to the tumor tissue, the glutathione content of the tumor was reduced by about 70% of its initial value. When administered into the tumors 2 hours prior to radiotherapy the animals so treated had a significantly longer median survival time compared with animals that received radiotherapy alone.

<p>The disease process giving rise to cancer involves the consecutive accumulation of genetic or genomic alterations impacting the normal regulation of cellular functions. In cases of hereditary cancers, this process may be stepwise, with a shared initiating lesion leading to common subsequent alterations. However, in many non-hereditary forms of cancer the initiating and subsequent alterations giving rise to the tumor can vary substantially from individual to individual, and multiple molecularly distinct subsets of the disease can exist within histopathologically similar tumors. This molecular heterogeneity between patients hinders the ability to identify which alterations are responsible for tumor development and subsequent maintenance, and confounds the ability to effectively treat patients as response to a particular therapeutic intervention may be highly dependent on the molecular composition of the disease.</p><p>To further our understanding of the molecular alterations associated with tumorigenesis, we analyzed aggressive brain cancer, glioblastoma (GBM), samples for which multiple types of genome-wide information was available. We utilized a series of in vitro or clinically derived gene expression signatures by comparing gene expression of samples based on whether a particular cellular signaling pathway was known to be active or inactive. Using these signatures for cellular signaling deregulation, we examined the association between various genomic alterations and the relative activity of each pathway, identifying alterations that were enriched within patients that harbored similar profiles of pathway activation. These analyses lead to the identification of numerous previously uncharacterized alterations in GBM, including the identification of a ubiquitin-like gene, UBL3, that was associated not only with pathway signaling, but was also associated with poor patient outcome, as well as response of GBM xenograft models to treatment with standard of care therapeutic agents.</p><p>Further, given that the challenges involved in analyzing clinical samples include development methods for timely analysis of genomic data, we have described a framework to utilize these genomic signatures in a prospective setting by incorporating a non-overlapping reference dataset of similar tumor samples. This methodology allows the examination of pathway signaling, as captured by the signature, to be run in real-time when only a single patient sample is analyzed, and has a high degree of fidelity to the results generated from retrospective analysis across multiple tumor types. Together these studies have provided a novel framework for identification of significant genomic alterations that impact pathway signaling, as well as moving providing the mechanisms to analyze genomic signatures in a robust manner that accounts for the challenges associated with the prospective clinical setting.</p> / Dissertation

Glioblastoma multiforme (GBM) is the most aggressive and most common primary brain tumor in adults accounting for 50-60% of primary brain tumors. The prognosis for patients with GBM remains poor and treatment is mainly palliative with a mean survival time of less than one year. Radiotherapy is used extensively in the management of glioblastoma either alone or in combination with surgery and/or chemotherapy. However, this tumor is one of the most resistant tumors to radiotherapy thus limiting the benefit of this form of treatment. <p>Studies have shown that malignant tumors have a high content of glutathione an antioxidant responsible for protecting the cells against damage from free radicals (mainly superoxide, hydroxyl and hydrogen peroxide). It is well established that glutathione, by neutralizing these free radicals plays a major role in radioresistance. Glioblastoma has relatively high levels of glutathione. In this study, by reducing the glutathione content of glioblastoma in a rat model, we were able to investigate the effect of this reduction in enhancing the effect of radiotherapy as a form of treatment for glioblastoma multiforme in a rat model. <p>By injecting L-Buthionine-SR-Sulfoximine (BSO) in to the tumor tissue, the glutathione content of the tumor was reduced by about 70% of its initial value. When administered into the tumors 2 hours prior to radiotherapy the animals so treated had a significantly longer median survival time compared with animals that received radiotherapy alone.

Aptamer probes for specific recognition of glioblastoma multiforme were generated using a repetitive and broad cell-SELEX-based procedure without negative selection. The 454 sequencing technology was used to monitor SELEX, and bioinformatics tools were used to identify aptamers from high throughput data. A group of aptamers were generated that can bind to target cells specifically with dissociation constants (K d ) in the nanomolar range. Selected aptamers showed high affinity to different types of glioblastoma cell lines, while showing little or no affinity to other cancer cell lines. The aptamers generated in this study have potential use in different applications, such as probes for diagnosis and devices for targeted drug delivery, as well as tools for molecular marker discovery for glioblastomas.

Η ΒΥΠ διαθέτει αντίτυπο της διατριβής σε έντυπη μορφή στο βιβλιοστάσιο διδακτορικών διατριβών που βρίσκεται στο ισόγειο του κτιρίου της. / Τα κακοήθη γλοιώματα συνιστούν τον πιο κοινό τύπο καρκίνου του κεντρικού νευρικού συστήματος στους ενήλικες και χαρακτηρίζονται από υψηλό βαθμό αγγειογένεσης και διήθησης στον παρακείμενο φυσιολογικό ιστό. Αν και οι θεραπευτικές προσεγγίσεις που χρησιμοποιούνται περιλαμβάνουν χειρουργική επέμβαση, ακτινοθεραπεία και χημειοθεραπεία, ποσοστό μεγαλύτερο του 90% των ασθενών εμφανίζουν υποτροπή της νόσου και η συνολική επιβίωση είναι εξαιρετικά χαμηλή. Τα παραπάνω γεγονότα εντείνουν την ανάγκη για την εφαρμογή πιο αποτελεσματικών θεραπευτικών σχημάτων, καθώς και την ανακάλυψη νέων μορίων-στόχων για τη θεραπεία του συγκεκριμένου τύπου καρκίνου. Η HARP είναι ένας εκκρινόμενος αυξητικός παράγοντας με μοριακό βάρος 18 kDa που έχει ισχυρή συγγένεια με την ηπαρίνη. Η HARP εκφράζεται κυρίως κατά την εμβρυϊκή ανάπτυξη και απομονώθηκε για πρώτη φορά ως πρωτεΐνη που προάγει την επέκταση των νευριτών στον αναπτυσσόμενο εγκέφαλο. Στην HARP έχει αποδοθεί ένας σημαντικός αριθμός βιολογικών δράσεων, όπως η συμμετοχή της στον πολλαπλασιασμό, στη μετανάστευση και στη διαφοροποίηση των κυττάρων, στο σχηματισμό οστών, στη χονδρογένεση, στη σπερματογένεση, στην ογκογένεση και στην αγγειογένεση. Η HARP εκφράζεται σε διάφορες καρκινικές σειρές από μηνιγγίωμα, νευροβλάστωμα, αστροκυττάρωμα, μελάνωμα, μικροκυτταρικό τύπο καρκίνου του πνεύμονα και γλοιοβλάστωμα. Κλινικές μελέτες έχουν αναφέρει αυξημένα επίπεδα της HARP στον ορό αίματος ασθενών με καρκίνο του κόλον του εντέρου, με καρκίνο του παγκρέατος και με καρκίνο του μαστού. Στην παρούσα μελέτη, προσπαθήσαμε να διερευνήσουμε το ρόλο της HARP στην ανάπτυξη όγκου και στην αγγειογένεση που προκαλούν τα κύτταρα C6, μετασχηματίζοντας τα κύτταρα αυτά με την αντινοηματική αλληλουχία για το cDNA της HARP και καταστέλλοντας την έκφρασή της στα κύτταρα C6. Μείωση της ενδογενούς έκφρασης της πρωτεΐνης της HARP στα κύτταρα C6 (κύτταρα AS-C6) προκάλεσε στατιστικά σημαντική αύξηση στον πολλαπλασιασμό, στην ανάπτυξη των κυττάρων σε τρισδιάστατο περιβάλλον και στη μετανάστευση. Όταν κύτταρα AS-C6 εμβολιάστηκαν σε χοριοαλλαντοϊκή μεμβράνη εμβρύου όρνιθας, οδήγησαν σε σημαντική αύξηση της καρκινικής αγγειογένεσης, σε σύγκριση με τις μεμβράνες που είχαν εμβολιαστεί με μη μετασχηματισμένα κύτταρα C6 ή με κύτταρα C6 που είχαν μετασχηματιστεί μόνο με το πλασμίδιο-φορέα (κύτταρα ΡC-C6). Με παρόμοιο τρόπο, θρεπτικό μέσο καλλιέργειας των κυττάρων AS-C6 προκάλεσε στατιστικά σημαντική αύξηση στον πολλαπλασιασμό, στη μετανάστευση και στο σχηματισμό ψευδαγγείων των ενδοθηλιακών κυττάρων in vitro σε σύγκριση με την επίδραση του θρεπτικού μέσου καλλιέργειας των κυττάρων C6 και ΡC-C6. Η έκφραση της HARP φαίνεται να είναι απαραίτητη για την ανασταλτική δράση αυξημένων ενδοκυτταρικών επιπέδων cAMP στον πολλαπλασιασμό και τη μετανάστευση των κυττάρων C6 και αύξηση των ενδοκυτταρικών επιπέδων cAMP οδήγησε σε αύξηση της έκφρασης και έκκρισης HARP, πιθανά μέσω ενεργοποίησης του μεταγραφικού παράγοντα AP-1. Τέλος, παρατηρήθηκε σημαντική αύξηση των επιπέδων του mRNA και της πρωτεΐνης του αυξητικού παράγοντα των ενδοθηλιακών κυττάρων (vascular endothelial growth factor, VEGF) σε καλλιέργειες των κυττάρων AS-C6 σε σχέση με τα κύτταρα C6 και PC-C6. H HARP βρέθηκε να συν-ανοσοκατακρημνίζεται με τον VEGF σε θρεπτικό μέσο καλλιέργειας των κυττάρων C6 και pC-C6, το οποίο υποδηλώνει μια άμεση αλληλεπίδραση μεταξύ των δύο αυτών αυξητικών παραγόντων. Συνοψίζοντας, τα αποτελέσματα της παρούσας εργασίας προτείνουν ότι ο αυξητικός παράγοντας HARP έχει αρνητικό ρόλο στη ρύθμιση της ανάπτυξη όγκου και της αγγειογένεση που επάγουν τα κύτταρα C6, πιθανά ρυθμίζοντας την έκφραση ή/και τη διαθεσιμότητα άλλων αυξητικών παραγόντων. / Malignant gliomas, the most common type of brain tumors in adults, present a remarkable degree of neovascularisation and invasiveness into surrounding tissues. Although combinations of surgery, radiotherapy and chemotherapy are used, more than 90% of the patients experience local recurrence and their survival is extremely low. These facts stress the need for more effective therapeutic strategies and new targets for tumour therapy. Heparin affin regulatory peptide (HARP), also known as pleiotrophin or heparin-binding growth-associated molecule, is an 18-kDa secreted growth factor that has high affinity for heparin. HARP is mainly expressed during embryonic development, in early postnatal rat and bovine brain and was first isolated as a major neurite outgrowth-promoting protein of developing brain. Since then, a number of biological activities have been well established for HARP, such as its role in cellular proliferation, migration and differentiation and its involvement in bone formation, chondrogenesis, spermatogenesis, tumor growth and angiogenesis. HARP is expressed in various cancer cell lines, derived from meningiomas, neuroblastomas, astrocytomas, melanomas, small cell lung cancer (SCLC) cell lines and glioblastomas. Clinical studies have shown elevated serum levels and tumor expression of HARP in patients with colon, stomach, pancreatic and breast cancer. Ιn the present study, we tried to elucidate the role of HARP in the growth and angiogenicity of C6 glioma cells by using an antisense strategy for inhibition of HARP expression in rat C6 glioma cells. Decrease of the expression of endogenous HARP protein in C6 cells (AS-C6 cells) significantly increased proliferation rate and anchorage-independent growth of cells. Implantation of AS-C6 cells onto chicken embryo chorioallantoic membranes resulted in a significant increase of tumor-induced angiogenesis, compared with those induced by non transfected or C6 cells transfected with the plasmid alone (PC-C6 cells). In the same line, conditioned medium from AS-C6 cells significantly increased endothelial cell proliferation, migration and tube formation in vitro compared with the effect of C6 or PC-C6 cells. HARP expression is required for cAMP-induced inhibition of C6 cell proliferation and migration and cAMP increased the expression and secretion of HARP by C6 cells, possibly through activation of Activator Protein 1. Finally, a significant increase in vascular endothelial growth factor (VEGF) mRNA and protein levels were observed in cultures of AS-C6 cells compared with C6 or PC-C6 cells. HARP was co-immunoprecipitated with VEGF from the conditioned medium of C6 and PC-C6 cells, which indicates a direct interaction between the two factors. Collectively, these data suggest that HARP negatively regulates tumor growth and angiogenesis induced by C6, possibly through regulation of the expression or/and activation of other growth factors.