<p>The incidence of urinary bladder carcinoma is increasing and many patients die every year of this disease despite assumed radical therapy. Thus, there is a need for improved methods of diagnosis and therapy. Radionuclide targeting is based on achieving specific delivery of radioactive nuclides to tumour cells with minimal damage to surrounding normal tissues. Two possible target structures are the epidermal growth factor (EGF) receptor and the related receptor HER-2.</p><p>Cellular binding and retention of ¹²⁵I-EGF-dextran conjugates was investigated in two bladder carcinoma cell lines. The conjugate bound specifically to the EGF receptor with delayed maximum binding, limited intracellular degradation and prolonged cellular retention compared to ¹²⁵I-EGF.</p><p>EGF was labelled using different radionuclides and methods. All the labelled variants bound specifically to the tumour cells although the cellular binding patterns and retention varied considerably. ¹¹¹In-DTPA-EGF had highest cellular retention and in decreasing order ²¹¹At-benzoyl-EGF and ¹²⁵I-labelled EGF.</p><p>Bladder cancer spheroids bound both ¹²⁵I-EGF-dextran as well as ¹²⁵I-EGF. Conjugate binding increased during a 48 h incubation period and was most prominent in the outer cell layers. The length of the dextran chain appeared not to alter the binding pattern.</p><p>The expression of EGF receptors and HER-2 in metastases and primary bladder carcinoma tumours was investigated. Both receptors were expressed in the majority of metastases and primary tumours.</p><p>Targeting the EGF receptor and/or HER-2 in urinary bladder carcinoma is an exciting new concept.</p>

<p>Tyrosine phosphorylation is a central mechanism in cellular signalling leading to proliferation, migration or differentiation. Protein tyrosine phosphorylation is regulated by the coordinated actions of protein tyrosine kinases and protein tyrosine phosphatases. This thesis investigates the involvement of tyrosine phosphatases in contact-induced growth inhibition of cells. Furthermore, it describes the structure and function of the extracellular domain of the receptor-like tyrosine phosphatase DEP- 1. </p><p>Tyrosine phosphatases, negatively regulating tyrosine kinases, have been suggested being involved in contact-induced growth inhibition of cells. Both endogenous EGF-receptors and transfected PDGF receptors showed a decreased ligand-induced tyrosine phosphorylation in cells of dense cultures. This difference was found to be due to increased receptor-directed tyrosine phosphatase activity in dense cultures. </p><p>Density enhanced tyrosine phosphatase-1 (DEP-1) is a receptor-like tyrosine phosphatase. It was found that DEP-1 contains chondroitin sulfate chains, thus identifying DEP-1 as a proteoglycan. Furthermore, DEP-1 was found to interact with a heparan sulfate proteoglycan. </p><p>No ligands have been identified for DEP-1. We have established a biacore-based assay for the identification of molecules interacting with the extracellular domain of DEP-1. A library of cell conditioned media was screened with the biacore assay. One of the samples was found to contain DEP-1 interacting molecules. Purification of the ligand has been initiated. </p><p>In an attempt to identify modulators of DEP-1 activity, Matrigell™ , a preparation of extracellular matrix was investigated. Stimulation with Matrigel™ was found to increase the specific activity of DEP-1 through interactions between the extracellular domain of DEP-1 and Matrigel™ component(s). </p>

<p>The DNA <i>double-strand break</i> (DSB) is a critical lesion which, if not completely restored, can have serious biological consequences. The <i>relative biological effectiveness</i> (RBE) of many severe end-points are closely related to radiation quality, with increased effectiveness at elevated ionization density. Data presented provide information about the influence of radiation quality on the initial processes causing DNA damage, and the mechanisms leading to its restoration. Such information will increase the understanding of radiation action mechanisms in mammalian cells. </p><p>Human cells were irradiated with accelerated ions having <i>linear energy transfer</i> (LET) values in the range 40-225 keV/μm, and ⁶⁰Co-photons. Detailed analyses of the DNA fragment distributions were performed in the size-range 5 kilobasepairs to 6 megabasepairs by pulsed-field gel electrophoresis. </p><p>A non-random fragmentation of DNA was evident, with an elevated number of small and medium-sized fragments for ion irradiation, and the total number of breaks increased by 80-110% when these fragments were included in the analyses. The RBE for DSB induction was 1.2-1.5. A two-fold increase of the number of breaks induced per nitrogen ion passing the cell nuclues was found when LET was increased from 80 to 225 keV/μm, indicating a possible role of particle track structure in DSB induction. Furthermore, the ability to repair DNA was closely related to radiation quality, with an increased proportion of unrejoined breaks for densely ionizing radiation. Surprisingly, the majority of breaks were rapidly rejoined even following exposure to high-LET radiation. The proportion of breaks restored by the slow phase showed a five-fold increase for the highest LET tested, compared with photons. The results presented nominates the complexity of breaks as one determining factor for reduced reparability reported following high-LET exposure.</p>

<p>Stem Cell Factor (SCF) can function as a survival factor, a mitogen or a chemoattractant depending on cell type. Binding of SCF to c-Kit induces dimerization and subsequent autophosphorylation of the receptor. This thesis describes the intracellular signal transduction elicited by c-Kit. </p><p>In the search for signal transduction molecules binding to activated c-Kit, we identified the adaptor proteins Grb2 and Grb7 as interacting partners. Grb2 could associate to Tyr-703 in the kinase insert as well as to Tyr-936 in the C-terminal tail of c-Kit. However, Grb7 could only bind to Tyr-936. </p><p>Tyr-568 in c-Kit is essential for SCF induced association and activation of Src family kinases (SFK). A mutated receptor that could not activate SFK (Y568F or Y568/570F) showed reduced Shc phosphorylation, Ras GTP loading, Erk activation and induction of <i>c-fos</i>. However, activation of SFK is not essential for the mitogenic response as measured by DNA synthesis.</p><p>Tyr-900 in c-Kit was identified as a SFK dependent phosphorylation site. The adaptor protein Crk and the p85 subunit of PI3’-kinase could associate with phosphorylated Tyr-900. In addition, we could demonstrate a constitutive complex between Crk and p85, suggesting indirect binding of Crk to Tyr-900 via p85. Mutation of Tyr-900 (Y900F) led to a reduced phosphorylation of Crk-II, loss of the second wave of Erk phosphorylation and a reduced mitogenic response. In addition the mutated receptor showed an increased ligand-induced degradation as compared to the wild-type receptor.</p><p>There exist two splice forms of c-Kit that differ in the presence or absence of four amino acids in the extracellular juxtamembrane region. These splice forms bind SCF with similar affinity but display striking differences in signaling characteristics, <i>e.g.</i> in phosphorylation kinetics, ligand-induced c-Kit degradation and activation of Erks. However, other pathways are activated similarly by both splice forms, such as the Ser/Thr kinase Akt which lies downstream of PI3’-kinase. In this study we show that differential phosphorylation of the various tyrosine residues occurs. Interestingly, Tyr-568 is more efficiently phosphorylated in the shorter form leading to stronger binding of SFK, whereas the PI3’-kinase binding site showed a similar degree of phosphorylation consistent with the data on Akt activation.</p>

The DNA double-strand break (DSB) is a critical lesion which, if not completely restored, can have serious biological consequences. The relative biological effectiveness (RBE) of many severe end-points are closely related to radiation quality, with increased effectiveness at elevated ionization density. Data presented provide information about the influence of radiation quality on the initial processes causing DNA damage, and the mechanisms leading to its restoration. Such information will increase the understanding of radiation action mechanisms in mammalian cells. Human cells were irradiated with accelerated ions having linear energy transfer (LET) values in the range 40-225 keV/μm, and 60Co-photons. Detailed analyses of the DNA fragment distributions were performed in the size-range 5 kilobasepairs to 6 megabasepairs by pulsed-field gel electrophoresis. A non-random fragmentation of DNA was evident, with an elevated number of small and medium-sized fragments for ion irradiation, and the total number of breaks increased by 80-110% when these fragments were included in the analyses. The RBE for DSB induction was 1.2-1.5. A two-fold increase of the number of breaks induced per nitrogen ion passing the cell nuclues was found when LET was increased from 80 to 225 keV/μm, indicating a possible role of particle track structure in DSB induction. Furthermore, the ability to repair DNA was closely related to radiation quality, with an increased proportion of unrejoined breaks for densely ionizing radiation. Surprisingly, the majority of breaks were rapidly rejoined even following exposure to high-LET radiation. The proportion of breaks restored by the slow phase showed a five-fold increase for the highest LET tested, compared with photons. The results presented nominates the complexity of breaks as one determining factor for reduced reparability reported following high-LET exposure.

The incidence of urinary bladder carcinoma is increasing and many patients die every year of this disease despite assumed radical therapy. Thus, there is a need for improved methods of diagnosis and therapy. Radionuclide targeting is based on achieving specific delivery of radioactive nuclides to tumour cells with minimal damage to surrounding normal tissues. Two possible target structures are the epidermal growth factor (EGF) receptor and the related receptor HER-2. Cellular binding and retention of 125I-EGF-dextran conjugates was investigated in two bladder carcinoma cell lines. The conjugate bound specifically to the EGF receptor with delayed maximum binding, limited intracellular degradation and prolonged cellular retention compared to 125I-EGF. EGF was labelled using different radionuclides and methods. All the labelled variants bound specifically to the tumour cells although the cellular binding patterns and retention varied considerably. 111In-DTPA-EGF had highest cellular retention and in decreasing order 211At-benzoyl-EGF and 125I-labelled EGF. Bladder cancer spheroids bound both 125I-EGF-dextran as well as 125I-EGF. Conjugate binding increased during a 48 h incubation period and was most prominent in the outer cell layers. The length of the dextran chain appeared not to alter the binding pattern. The expression of EGF receptors and HER-2 in metastases and primary bladder carcinoma tumours was investigated. Both receptors were expressed in the majority of metastases and primary tumours. Targeting the EGF receptor and/or HER-2 in urinary bladder carcinoma is an exciting new concept.

Tyrosine phosphorylation is a central mechanism in cellular signalling leading to proliferation, migration or differentiation. Protein tyrosine phosphorylation is regulated by the coordinated actions of protein tyrosine kinases and protein tyrosine phosphatases. This thesis investigates the involvement of tyrosine phosphatases in contact-induced growth inhibition of cells. Furthermore, it describes the structure and function of the extracellular domain of the receptor-like tyrosine phosphatase DEP- 1. Tyrosine phosphatases, negatively regulating tyrosine kinases, have been suggested being involved in contact-induced growth inhibition of cells. Both endogenous EGF-receptors and transfected PDGF receptors showed a decreased ligand-induced tyrosine phosphorylation in cells of dense cultures. This difference was found to be due to increased receptor-directed tyrosine phosphatase activity in dense cultures. Density enhanced tyrosine phosphatase-1 (DEP-1) is a receptor-like tyrosine phosphatase. It was found that DEP-1 contains chondroitin sulfate chains, thus identifying DEP-1 as a proteoglycan. Furthermore, DEP-1 was found to interact with a heparan sulfate proteoglycan. No ligands have been identified for DEP-1. We have established a biacore-based assay for the identification of molecules interacting with the extracellular domain of DEP-1. A library of cell conditioned media was screened with the biacore assay. One of the samples was found to contain DEP-1 interacting molecules. Purification of the ligand has been initiated. In an attempt to identify modulators of DEP-1 activity, Matrigell™ , a preparation of extracellular matrix was investigated. Stimulation with Matrigel™ was found to increase the specific activity of DEP-1 through interactions between the extracellular domain of DEP-1 and Matrigel™ component(s).

Stem Cell Factor (SCF) can function as a survival factor, a mitogen or a chemoattractant depending on cell type. Binding of SCF to c-Kit induces dimerization and subsequent autophosphorylation of the receptor. This thesis describes the intracellular signal transduction elicited by c-Kit. In the search for signal transduction molecules binding to activated c-Kit, we identified the adaptor proteins Grb2 and Grb7 as interacting partners. Grb2 could associate to Tyr-703 in the kinase insert as well as to Tyr-936 in the C-terminal tail of c-Kit. However, Grb7 could only bind to Tyr-936. Tyr-568 in c-Kit is essential for SCF induced association and activation of Src family kinases (SFK). A mutated receptor that could not activate SFK (Y568F or Y568/570F) showed reduced Shc phosphorylation, Ras GTP loading, Erk activation and induction of c-fos. However, activation of SFK is not essential for the mitogenic response as measured by DNA synthesis. Tyr-900 in c-Kit was identified as a SFK dependent phosphorylation site. The adaptor protein Crk and the p85 subunit of PI3’-kinase could associate with phosphorylated Tyr-900. In addition, we could demonstrate a constitutive complex between Crk and p85, suggesting indirect binding of Crk to Tyr-900 via p85. Mutation of Tyr-900 (Y900F) led to a reduced phosphorylation of Crk-II, loss of the second wave of Erk phosphorylation and a reduced mitogenic response. In addition the mutated receptor showed an increased ligand-induced degradation as compared to the wild-type receptor. There exist two splice forms of c-Kit that differ in the presence or absence of four amino acids in the extracellular juxtamembrane region. These splice forms bind SCF with similar affinity but display striking differences in signaling characteristics, e.g. in phosphorylation kinetics, ligand-induced c-Kit degradation and activation of Erks. However, other pathways are activated similarly by both splice forms, such as the Ser/Thr kinase Akt which lies downstream of PI3’-kinase. In this study we show that differential phosphorylation of the various tyrosine residues occurs. Interestingly, Tyr-568 is more efficiently phosphorylated in the shorter form leading to stronger binding of SFK, whereas the PI3’-kinase binding site showed a similar degree of phosphorylation consistent with the data on Akt activation.

(PI3K)/AKT signaling pathway could be a cause of therapeutic resistance in breast cancer. The PI3K/AKT pathway controls cell proliferation, cell growth and survival, and its members include oncogenes and tumor suppressor genes. Alterations in this pathway are frequent in cancer. In this thesis, we aimed to study the biological significance of some of these alterations in a tumor context as well as their clinical value. PIK3CA gene, encoding the PI3K catalytic subunit, was examined for mutations. The tumor suppressor PTEN, that counteracts PI3Kmediated effects, was studied at the protein level whereas amplification of RPS6KB1 (S6K1) and RPS6KB2 (S6K2) genes, encoding two substrates of the mammalian target of rapamycin (mTOR) acting downstream PI3K/AKT, was also inspected. AKT phosphorylation or activation (pAKT) was determined by immunohistochemistry. Other factors related with this pathway, such as HER-2, heregulin (HRG) β1, the cell cycle inhibitor p21WAF1/CIP1, the pro-apoptotic factor Bcl-2, and cyclin D1,  were also considered. These studies were perfomed in two patient materials consisting of premenopausal patients that received endocrine treatment (paper I) and postmenopausal patients randomized to receive radiotherapy (RT) or chemotherapy (CMF) in combination with tamoxifen (Tam) or no endocrine treatment (papers II-IV). In the first material, we found that pAKT indicated higher risk of distant recurrence among endocrine treated patients. In the second material HRGβ1 induced accumulation cytoplasmic p21 in vitro and pAKT was associated with cytoplasmic p21 in the tumors. In addition, p21 cellular location identified subgroups of ER+ patients with different responses to tamoxifen. Other alterations such as PIK3CA mutations and PTEN loss were positively associated in this material. PIK3CA mutations lowered the risk for local recurrences while PTEN loss conferred radiosensitivity as a single variable or combined with mutated PIK3CA. PIK3CA mutations and/or PTEN loss was associated with lower S-phase (SPF). Nevertheless, among patients with low proliferating tumors, these alterations predicted higher risk of recurrence in contrast to those with high proliferating tumors. Finally, we found amplification of the S6K1 and S6K2 genes. S6K2 amplification was associated with cyclin D1 gene amplification, predicted poor recurrence-free survival and breast cancer death, and indicated benefit from tamoxifen. On the other hand, S6K1 amplification was associated with HER-2 amplification/overexpression, indicated higher risk of recurrence and was a predictor of poor response to radiotherapy. These results indicate the potential of this pathway as therapeutic source. / Bröstcancer är en vanlig sjukdom och dödsorsak bland kvinnor i Sverige. Könshormonet östrogen tillsammas med cellernas receptorer för hormonet spelar en viktig roll för bröstcancerutvecklingen. Därför behandlas denna sjukdom med anti-hormonella substanser inriktade mot hämning av östrogensyntes/östrogen receptorn. Tamoxifen är den vanligaste formen av anti-östrogenbehandling som används efter operation. Tamoxifenbehandling förbättrar betydligt 5-årsöverlevnaden hos patienter med östrogenreceptorpositiva tumörer. Emellertid finns det patienter som återkommer med metastaser efter en tid. I det här projektet studerar vi andra receptorer samt deras signalvägar som kan aktivera östrogenreceptorn och därmed orsaka tamoxifenresistens. En sådan receptor är HER-2 vilken överuttrycks i 15-20% vid bröstumörer. HER-2 receptorn kan rekrytera proteiner med enzymatisk aktivitet, till exempel PI3K. PI3K aktiverar ett annat enzym, AKT, vilket är inblandat i en kaskad som leder till tumörtillväxt och tumöröverlevnad (genom till exempel aktivering av östrogenreceptorn). Våra resultat hitills visar att patienter med aktiverat AKT (pAKT) har större risk att få metastaser och därmed sämre överlevnad än patienter utan pAKT, detta trots hormonell behandling. I större material där HER-2 proteinuttrycket korrelerar med pAKT har vi också funnit att patienter med AKTnegativa tumörer kunde dra nytta av både tamoxifen och strålbehandling. Vi har även undersökt PIK3CA genen (som kodar för en del av PI3K) och hittat mutationer i 24% av bröstumörerna. Det är dock ännu oklart hur dessa mutationer ska tas hänsyn till för att kunna bestämma en effektiv behandling. PTEN är ett annat enzym som motverkar PI3K-aktivitet. Bortfall av PTEN förekommer ofta i bröstcancer och  har associerats med PI3K/AKT aktivering. I vårt material var PTEN-förlust frekvent (37%) och associerades med PIK3CA mutationer. PTEN förlust som ensam faktor eller tillsammans med PIK3CA mutationer ökade strålkänslighet. Andra proteiner som är inblandade i PI3K signalvägen är S6K1 och S6K2 och dessa har betydelse för cellens proteinsyntes. Nyligen har vi kunnat visa att generna för både S6K1/2 finns i många kopior (genamplifering) I tumörcellerna hos bröstcancerpatienter. Dessutom fanns det ett positivt samband mellan S6K1/2 amplifiering och amplifiering av andra kända cancergener (som t. ex HER-2 och cyclin D1) men förhållandet till PIK3CA-mutationer var det omvända. Patienter med antigen S6K1 eller HER-2 amplifierade tumörer svarade dåligt på strålbehandling men skulle möjligen kunna behandlas med en specifik substans riktad mot S6K1 eller HER-2. Ett ökat antal kopior av S6K2 indikerade dålig prognos men bra nytta av tamoxifen. Våra resultat visar att PI3K/AKT signalvägen ofta är aktiverad vid bröstcancer och skulle kunna vara en viktig måltavla för behandling.

To improve outcome in young, high-risk lymphoma patients, treatment was intensified, adding etoposide and rituximab to standard CHOP treatment. Granulocyte-colony stimulating factor (G-CSF) enabled treatment bi-weekly. Results were promising: overall (OS) and event-free survival (EFS) 79% and 60% respectively, median follow up 27 months. Single infusion Ara-C, contrary to expectations, did not prevent relapse in CNS. DLBCL were classified as germinal center (GC) or non-GC derived, using immunohistochemical markers, CD10, BCL6 and MUM1. We investigated the outcome for both phenotypes after adding rituximab to chemotherapy. For 106 patients treated with CHOP alone, the GC phenotype displayed significantly better OS and EFS. In contrast, GC phenotype did not predict outcome in 95 patients treated with immunochemotherapy . Thus, addition of rituximab seems to eliminate the prognostic value of immunohistochemically defined GC phenotypes in DLBCL. To improve evaluation and find non-responders, mid-treatment FDG-PET CT was incorporated into clinical routine for patients with high-risk aggressive lymphoma. For those with positive PET, biopsy followed by treatment intensification was recommended. Twenty-five patients were examined, five with positive PET. Two of these had lymphoma in the biopsy. Two had a negative biopsy, and one had a false positive investigation. Seven patients had increased uptake of uncertain significance. Two patients with uncertain PET, and two with negative PET have relapsed, giving a negative predictive value of 85%. In case of relapse of aggressive lymphoma or if not obtaining CR, high dose chemotherapy with autologous stem cell support (HDT) is standard treatment. HDT outcome for 38 patients with transformed follicular lymphoma was compared to outcome for 79 patients with de novo B-cell lymphoma. At median follow-up of 11.5 years both OS and EFS were superior in the transformed group, OS 67% and 33%, EFS 55% and 27% respectively. Treatment related mortality was less than reported in other studies.