Malignant cells increase glycolysis and down regulate mitochondrial respiration for ATP production. Mechanisms for respiratory impairment in cancerous cells and their importance for carcinogenesis are not well defined. We found that expression of the respiration-inducing uncoupling protein 3 (UCP3) was normally expressed in murine skin and was greatly decreased in cutaneous malignancies. To better understand the significance of UCP3 in epidermal biology and to test the importance of respiratory changes in cancer development, we generated hemizygous mice expressing a keratin-5 promoter-UCP3 transgene (K5-UCP3). Compared to wild type, K5-UCP3 mice exhibited increased cutaneous mitochondrial respiration, had decreased mitochondrial membrane potential in isolated keratinocytes, and were completely resistant to chemically-induced skin carcinogenesis. We showed that the mechanism of UCP3-dependent cancer protection is most likely not due to increased intracellular heat production or ATP depletion in pre-cancerous cells. Therefore, because hair follicle "bulge" stem cells (bSC) are K5⁺ and progenitors of cutaneous carcinomas, we hypothesized that K5-UCP3 animals were protected from skin carcinogenesis due to alterations in their bSC population. Unlike WT, most (85%) hair follicle bulge regions in K5-UCP3 mice lost biochemical markers of quiescent bSC, but bSC functions were fully intact. Supporting our hypothesis that increased skin turnover protected K5-UCP3 mice from skin cancer; we showed that basal keratinocyte cell cycling was increased 3% in K5-UCP3 skin compared to WT. Moreover, the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induced similar proliferative responses in both WT and K5-UCP3 skin, but the magnitude of TPA-induced skin thickening was greatly decreased in K5-UCP3 versus WT mice. Together with microarray, histochemical and in vitro morphologic analyses showing that keratinocyte differentiation was sharply increased in K5-UCP3 skin, this implies that UCP3 may increase keratinocyte transit from stem to differentiated daughter cells. Thus, the cancer resistance mechanism in K5-UCP3 mice likely stems from UCP3-induced mitochondrial respiration, which promotes the differentiation and abrogates the tumorigenicity of progenitor keratinocytes. This is the first demonstration in any context that UCP3 blocks carcinogenesis and promotes cellular differentiation. These observations support Warburg's contention that respiratory dysfunction promotes cancer development, and suggest that mitochondrial uncoupling may be a novel target for cancer prevention and treatment. / text

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Mutation or inactivation of RB occurs in most human tumors and results in the deregulation of several E2F family transcription factors. Among the E2F family, E2F3 has been implicated as a key regulator of cell proliferation and E2f3 gene amplification and overexpression is detected in some human tumors. To study the role of E2F3a in tumor development, we established a transgenic mouse model expressing E2F3a in a number of epithelial tissues via a keratin 5 (K5) promoter. Transgenic expression of E2F3a leads to hyperproliferation, hyperplasia and increased levels of p53-independent apoptosis in transgenic epidermis. Consistent with data from human cancers, the E2f3a transgene is found to have a weak oncogenic activity on its own and to enhance the response to a skin carcinogenesis protocol. While E2F3a induces apoptosis in the absence of p53, the inactivation of both p53 and p73, but not p73 alone, significantly impairs apoptosis induced by E2F3a. This suggests that both p53 and p73 contribute to E2F3a induced apoptosis but that their function is compensatory. Even though data suggest that E2F3a carries out its unique apoptotic activity in part through another E2F family member E2F1, unlike E2F1, the ARF tumor suppressor is required for E2F3a-induced apoptosis. While both E2F3a and E2F1 require ATM for apoptosis, E2F3a activates ATM through a distinct mechanism from E2F1. The overexpression of E2F3a results in the accumulation of DNA damage in K5 transgenic keratinocytes and normal human fibroblasts (NHFs). In response to this, the DNA damage checkpoint kinase ATM is activated, and phosphorylation of the downstream targets p53 and the histone variant H2AX are significantly increased. Additional studies show that increased Cdk activity and aberrant DNA replication contributes to DNA damage, ATM activation and apoptosis in response to deregulated E2F3a, which suggest that aberrant replication imposed by deregulated E2F3a plays an important role in the activation of the ATM DNA damage response pathway. Activation of ATM by E2F3a is not affected by loss of ARF or E2F1. Meanwhile, E2F3a-induced ARF upregulation is not affected by E2F1 loss. The above results indicate that E2F3a engages several parallel pathways involving E2F1, ARF and the ATM kinase, and these pathways cooperate to promote apoptosis.

published_or_final_version / Biochemistry / Master / Master of Philosophy

Τα καρκινώματα του λάρυγγα και του λαρυγγοφάρυγγα αποτελούν τη συχνότερη κακοήθη νεοπλασία Κεφαλής & Τραχήλου. Οι προκαρκινικές βλάβες είναι ενδιάμεσοι σταθμοί της καρκινογένεσης, που καταλήγει στον καρκίνο. Παράλληλα με την καρκινογένεση συμβαίνει ενεργοποίηση κυττάρων του υποεπιθηλιακού στρώματος. Η χημειοπρόληψη είναι απόπειρα φαρμακολογικής ανακοπής της καρκινογένεσης. Ο πυρηνικός υποδοχέας PPARγ καταλήγει σε διαφοροποίηση αρκετών κυττάρων. Ο RXRα ετεροδιμερίζεται με τον PPARγ και άλλους πυρηνικούς υποδοχείς. Ο μεταγραφικός παράγοντας NF-κB ενισχύει την κυτταρική επιβίωση και φλεγμονή. Ο μεμβρανικός υποδοχέας EGFR κινητοποιεί μιτογόνες διαδικασίες. Η COX-2 συνθέτει προσταγλανδίνες. Κατά την παρούσα εργασία, μελετήθηκε με ανοσοϊστοχημεία σε τομές παραφίνης η έκφραση των πέντε μορίων στο φυσιολογικό λαρυγγικό επιθήλιο, προκαρκινικές βλάβες και καρκινώματα διαφόρων Grade, καθώς και το υποκείμενο στρώμα, σε ιστικά δείγματα λάρυγγα από 127 ασθενείς. Από τους PPARγ, NF-κB(υπομονάδα p65) και RXRα, που εμφάνισαν μεικτή υποκυττάρια εντόπιση, οι δύο πρώτοι αναγνωρίστηκαν κυρίως στο κυτταρόπλασμα και ο τρίτος στον πυρήνα. Οι EGFR και COX-2 παρουσίασαν μεμβρανική και κυτταροπλασματική ανοσοδραστικότητα αντίστοιχα. Η έκφραση του υποδοχέα PPARγ συσχετίστηκε θετικά με την καλή διαφοροποίηση των όγκων. Ο RXRα ελαττώθηκε κατά την καρκινογένεση. Τα επίπεδα των παραγόντων NF-κB, EGFR και COX-2 παρουσίασαν αύξηση στην καρκινογένεση. Η COX-2 συσχετίστηκε αρνητικά με το Grade των όγκων. Στις υπόλοιπες συσχετίσεις μελετήθηκε η συνέκφραση των παραγόντων σε κάθε ασθενή. Παρατηρήθηκε στο φυσιολογικό και προνεοπλασματικό επιθήλιο, θετική σχέση της COX-2 με τους υπόλοιπους παράγοντες. Επιβεβαιώθηκε η συνέργεια των PPARγ & RXRα, ως προς την πρόκληση διαφοροποίησης στα καρκινικά κύτταρα. Διαπιστώθηκε κατασταλτική επίδραση του PPARγ στην καρκινογόνο δράση του NF-κB. Αποκαλύφθηκε συνεργιστική δράση της COX-2 στην PPARγ-εξαρτώμενη διαφοροποίηση, οφειλόμενη πιθανόν σε προσφορά της 15d-PGJ2, από το ένζυμο στον πυρηνικό υποδοχέα. Κατά την εκτίμηση του μικροπεριβάλλοντος, αξιολογήθηκε η έκφραση σε συνάρτηση με την ύπαρξη καρκινώματος και το βαθμό διαφοροποίησής του. Οι μυοϊνοβλάστες που περιβάλλουν καρκινώματα (CAFs), παρουσιάζουν επαγωγή των RXRα, NF-κB και COX-2. Μάλιστα η υψηλή έκφραση COX-2 στους CAFs, συνδυάζεται με υψηλό Grade. Ενεργοποίηση στους CAFs, συνδέεται με υπερ-δραστήρια NF-κB σηματοδότηση στο καρκίνωμα. Ο NF-κB υπερεκφράζεται στα λεμφοκύτταρα που διηθούν τους όγκους (TILs). Επίσης τα μακροφάγα γύρω από νεοπλάσματα (TAMs), περιέχουν υψηλό PPARγ. Η χρήση αγωνιστών των PPARγ, RXRα, με παράλληλη αναστολή των NF-κB, EGFR και COX-2, θα είχε πιθανότατα ευνοϊκό αποτέλεσμα στην αναστροφή της λαρυγγικής καρκινογένεσης. Το χημειοπροληπτικό σχήμα, θα ήταν ωφέλιμο να τροποποιείται ύστερα από εξατομικευμένη αξιολόγηση του δικτύου των πέντε παραγόντων. / Carcinomas of the larynx and laryngopharynx are the most common malignancies of the Head&Neck. The precancerous lesions are mid-points of carcinogenesis, which results in cancer. Carcinogenesis is accompanied by reactive initiation of stromal cells. Chemoprevention pursues the arrest of carcinogenesis, by pharmacological means. PPARγ, a nuclear receptor, promotes cellular differentiation. The nuclear receptor RXRα partners with PPARγ or other members of the superfamily. The transcription factor NF-κB enhances cell survival and inflammation. The receptor EGFR receives growth signals. The enzyme COX-2 perpetuates inflammation by means of prostaglandin synthesis. The present study, utilizing paraffin section immunohistochemistry, assessed expression of the five molecules in normal laryngeal epithelium, precancerous lesions and carcinomas, along with their adjacent stroma. Clinical samples were derived from 127 patients who had undergone biopsy or laryngectomy. Of the three molecules demonstrating mixed subcellular presence, PPARγ and NF-κB(p65 subunit) localized more frequently in cytoplasm, whereas RXRα expression was mainly nuclear. EGFR and COX-2 staining patterns were membranous and cytoplasmic, respectively. PPARγ correlated with high tumor differentiation. RXRα was diminished in dysplasia and cancer. NF-κB, EGFR and COX-2, were upregulated as tumorigenesis progressed. COX-2 showed an inverse relationship with tumor Grade. The remaining correlations are based on coexpression analysis of the aforementioned factors. COX-2 was positively associated in normal and preneoplastic epithelia with the four regulating proteins. The data verify the synergistic effect of PPARγ and RXRα, regarding potentiation of neoplastic cell differentiation. Suppressive influence of PPARγ on NF-κB–mediated carcinogenesis was manifested. COX-2 overexpression was shown to reinforce the beneficial role of PPARγ in cell differentiation, probably due to production of the receptor agonist 15d-PGJ2. Subepithelial stroma was examined with regard to immunoreactivity in relation to the presence of invasion as well as to tumor Grade. Carcinoma Associated Myofibroblasts (CAFs), manifested upregulation of RXRα, NF-κB and COX-2, which mediate in concert angiogenesis, inflammation, and tumor spread. Intense COX-2 expression in CAFs, correlated with poor tumor differentiation. CAF activation was associated with intense NF-κB signalling in cancer cells. NF-κB was overexpressed in Tumor Infiltrating Lymphocytes (TILs). Finally, in Tumor Associated Macrophages (TAMs), PPARγ was induced. PPARγ, RXRα agonists, combined with NF-κB, EGFR and COX-2 inhibitors, would likely restrain laryngeal carcinogenesis. Subtle variations in the chemopreventive regimen, based on personalized molecular profiling, would hopefully achieve a patient-tailored therapeutic approach.

Predictors of response of AIDS-associated Kaposi-Sarcoma to standard chemotherapy Overview: Kaposi Sarcoma is the most common HIV-associated cancer. Its etiology and pathogenesis is not fully understood. Little is known about what predicts prognosis, survival and therapeutic response in HIV-KS. In South Africa given the high seroprevalence rates of HIV-l and human herpes virus 8 (HHV 8), Kaposi's sarcoma is a significant problem. The majority of patients have been treated solely with palliation due to the poor outcome associated with a diagnosis of HIV-KS, more so in the absence of highly active antiretroviral therapy (HAART). Since the national ARV rollout programme and the availability and accessibility of HAART to all patients with a diagnosis of HIV-KS, a new strategy has to be established to enable adequate patient selection for chemotherapy. There have been a few published studies addressing the predictors of response to chemotherapy in the first world. However, this is the first study of these factors in HIV-l infected African patients with Kaposi's sarcoma. Aim: To identify and assess the potential value of several parameters predictive of outcome, survival and therapeutic response in HIV- infected patients with KS. Clinical, hematological, biochemical, immunological and virological variables were evaluated. Methods: We collected data from 25 patients with AIDS-KS who were enrolled in a phase III randomized controlled trial comparing HAART alone with the combination of HAART and chemotherapy. All patients were from the combination therapy arm. The following variables were evaluated as predictors of prognosis and therapeutic response: age, gender, ethnic origin, Haemoglobin (Hb), white blood cells (WBCs), lymphocytes, neutrophils, platelets, S.albumin, ALP, GGT, CD4 count, HIV viral load. These variables were assessed in patients at baseline and month 6 of therapy. Patients were staged into good risk and poor risk according to the AIDS clinical trial group (ACTG) criteria. The outcomes assessed were response to treatment and mortality. Results: A total of 25 patients participated to the study. Of these 16(64%) were males and 9(36%) were females, with male: female ratio of 2.7:1. Median age was 34 years (24-47); all patients were of Black African origin. Of the 21 patients, 15 (71.4%) were of good prognosis and 6(28.6%) were of poor prognosis. At baseline the median values of the different variables were as follows: Hb 10.9 g/dl, WBCs 5.95x109/L, lymphocytes 1.7 x109/L, neutrophils 3 x10 9 /L, platelets 272 x10 9 /L, S.albumin 30 gil, total protein 88 gil, ALP 64 U/L, and GTT 21 U/L, CD4 count was 255 cells/mm 3 , HIV-RNA viral load was 42000( 4.610gs). At month 6, 22 patients remained alive, their median values were: Hb 12.2 g/dl, WBCs 4.65 x109/L, lymphocytes 1.5 x109/L, neutrophils 3 x10 9 /L, platelets 301 x109/L, S.albumin 36.5 gil, total protein 84.5 gil, ALP 78.5 U/L, GTT 44.5 U/L, CD4 count 288 cells/mm3 , HIV-RNA viral load was 50500( 4.6910gs). The baseline median CD4 and HIV-RNA viral load counts for the 3 patients who died before month 6 were 47 cells/mm3 and 31000(4.610gs); respectively. Response to therapy was evaluated in 21(84%) patients as 4(16%) patients were missing, of the 21 patients 3 (14.3%) had complete response and 18(85.7%) had partial response. With respect to sex 2(14.3%) males had complete response and 12(85.7%) had partial response, 1(14.3%) female had complete response and 6 (85.7%) had partial response. Non-parametric statistics were used because of the small sample size and the skewness of the data. Variables were described using medians and ranges, and compared between two independent groups using Mann-Whitney tests. Baseline and month 6 comparisons were done using Wilcoxon signed ranks tests. Receiver Operating Characteristic (ROC) curves were used to analyze cut points to optimize sensitivity and specificity of a quantitative variable for a dichotomous outcome. Discussion In the univariate analysis age and sex didn't influence prognosis and therapeutic response, the influence of ethnic origin couldn't be assessed as all patients were of the same ethnic origin. Baseline WBCs (P= 0.004) and lymphocytes (P=0.026) were significantly associated with complete response. Higher values of GGT (p=O.OOl); ALP (P=0.006) were associated with more deaths. Baseline CD4 count and HIV viral load were not of predictive value, lthough change CD4 (P=002) and VL (p=.OOO) over time was significant and most likely attributed to response to therapy. 90.9 % of patients reached undetectable HIV-l Viral loads at month 6. CONCLUSION: Neither CD4 count nor HIV viral load at baseline predicted prognosis or survival; however there was a borderline significance of CD4 (P=0.058) towards a better survival. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2006.

This thesis consists of three main parts. An introduction to diode-pumped solid-state lasers, thermal modelling of solid-state lasers and rate-equation modelling of solid-state lasers. The first part explains the basic components and operation principles of a typical diode-end-pumped solid-state laser. The stimulated emission process, solid-state laser gain media, various pump geometries and a basic end-pumped laser resonator configuration are among the topics that are explained. Since thermal effects are one of the main limiting factors in the power-scaling of diode-pumped solid-state lasers, the second part of this thesis describes numerical and analytical thermal models that determine the thermal lens and thermally induced stresses in a laser crystal. As a first step, a time-independent numerical thermal model which calculates the three-dimensional temperature distribution in the laser crystal is implemented. In order to calculate the time dependent thermally induced stresses in a laser crystal, a coupled thermal-stress finite element analysis model was implemented. Even though some steady-state analytical solutions for simple crystal geometries do exist, the finite element analysis approach was taken so that the time dependent thermally induced stresses could be calculated for birefringent crystals of various geometries. In order to validate the numerical results, they are compared to experimental data and analytical solutions where possible. In the last part, the population dynamics inside the laser gain medium are described and modelled with a quasi-three-level rate-equation model. A comprehensive spatially resolved rate-equation model is developed and discussed. In order to simplify the implementation of the rate-equation model as a computer simulation, the spatial dependence of the laser parameters is ignored so that the model reduces to a singleelement plane-wave model. The simplified rate-equation model is implemented and solved numerically. The model is applied to a four-level CW and Q-switched Nd:YLF laser as well as a quasi-three-level QCW Tm:GdV04 laser. The models' predictions are thoroughly verified with experimental results and also with analytical solutions where possible. / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Durban, 2007.

Das Selenoprotein Glutathionperoxidase 2 (GPx2) ist ein epithelzellspezifisches, Hydroperoxide-reduzierendes Enzym, welches im Darmepithel, vor allem in den proliferierenden Zellen des Kryptengrundes, exprimiert wird. Die Aufrechterhaltung der GPx2-Expression im Kryptengrund auch bei subadäquatem Selenstatus könnte darauf hinweisen, dass sie hier besonders wichtige Funktionen wahrnimmt. Tatsächlich weisen GPx2 knockout (KO)-Mäuse eine erhöhte Apoptoserate im Kryptengrund auf. Ein Ziel dieser Arbeit war es deshalb, die physiologische Funktion der GPx2 näher zu untersuchen. In Kryptengrundepithelzellen aus dem Colon selenarmer GPx2 KO-Mäuse wurde eine erhöhte Caspase 3/7-Aktivität im Vergleich zum Wildtyp (WT) festgestellt. Zudem wiesen diese Zellen eine erhöhte Suszeptibilität für oxidativen Stress auf. Die GPx2 gewährleistet also den Schutz der proliferierenden Zellen des Kryptengrundes auch bei subadäquater Selenversorgung. Des Weiteren wurde im Colon selenarmer (-Se) und -adäquater (+Se) GPx2 KO-Mäuse im Vergleich zum WT eine erhöhte Tumornekrosefaktor α-Expression und eine erhöhte Infiltration von Makrophagen festgestellt. Durch Fütterung einer selensupplementierten Diät (++Se) konnte dies verhindert werden. In GPx2 KO-Mäusen liegt demnach bereits basal eine niedriggradige Entzündung vor. Dies unterstreicht, dass GPx2 vor allem eine wichtige antiinflammatorische Funktion im Darmepithel besitzt. Dem Mikronährstoff Selen werden protektive Funktionen in der Colonkanzerogenese zugeschrieben. In einem Mausmodell der Colitis-assoziierten Colonkanzerogenese wirkte GPx2 antiinflammatorisch und hemmte so die Tumorentstehung. Auf der anderen Seite wurden jedoch auch prokanzerogene Eigenschaften der GPx2 aufgedeckt. Deshalb sollte in dieser Arbeit untersucht werden, welchen Effekt ein GPx2 knockout in einem Modell der sporadischen, durch Azoxymethan (AOM) induzierten, Colonkanzerogenese hat. Im WT kam es in präneoplastischen Läsionen häufig zu einer erhöhten GPx2-Expression im Vergleich zur normalen Darmmucosa. Eine derartige Steigerung der GPx2-Expression wurde auch in der humanen Colonkanzerogenese beschrieben. Das Fehlen der GPx2 resultierte in einer verminderten Entstehung von Tumoren (-Se und ++Se) und präneoplastischen Läsionen (-Se und +Se). Somit förderte GPx2 die Tumorentstehung im AOM-Modell. Acht Stunden nach AOM-Gabe war im GPx2 KO-Colon im Vergleich zum WT eine erhöhte Apoptoserate in der Kryptenmitte (-Se, +Se), nicht jedoch im Kryptengrund oder in der ++Se-Gruppe zu beobachten. Möglicherweise wirkte GPx2 prokanzerogen, indem sie die effiziente Elimination geschädigter Zellen in der Tumorinitiationsphase verhinderte. Eine ähnliche Wirkung wäre auch durch die erhöhte GPx2-Expression in der Promotionsphase denkbar. So könnte GPx2 proliferierende präneoplastische Zellen vor oxidativem Stress, Apoptosen, oder auch der Antitumorimmunität schützen. Dies könnte durch ein Zusammenwirken mit anderen Selenoproteinen wie GPx1 und Thioredoxinreduktasen, für die ebenfalls auch prokanzerogene Funktionen beschrieben wurden, verstärkt werden. Eine wichtige Rolle könnte hier die Modulation des Redoxstatus in Tumorzellen spielen. Die Variation des Selengehalts der Diät hatte im WT einen eher U-förmigen Effekt. So traten in der –Se und ++Se-Gruppe tendenziell mehr und größere Tumore auf, als in der +Se Gruppe. Zusammenfassend schützt GPx2 also die proliferierenden Zellen des Kryptengrundes. Sie könnte jedoch auch proliferierende transformierte Zellen schützen und so die sporadische, AOM-induzierte Colonkanzerogenese fördern. In einem Modell der Colitis-assoziierten Colonkanzerogenese hatte GPx2 auf Grund ihrer antiinflammatorischen Wirkung einen gegenteiligen Effekt und hemmte die Tumorentstehung. Die Rolle der GPx2 in der Colonkanzerogenese ist also abhängig vom zugrunde liegenden Mechanismus und wird maßgeblich von der Beteiligung einer Entzündung bestimmt. / The selenoprotein glutathione peroxidase 2 (GPx2) is a hydroperoxide-reducing enzyme that is mainly expressed in the gastrointestinal epithelium, especially in the crypt base were the proliferating cells reside. GPx2 expression is maintained even when the selenium supply is limited, which indicates that GPx2 might have an important function in these cells. Indeed, GPx2 knockout (KO)-mice have an enhanced rate of apoptosis in the crypt base. Therefore one aim of this study was to further elucidate the physiological function of the GPx2. Isolated colonic crypt base epithelial cells of selenium deficient GPx2 KO-mice were found to have a higher caspase 3/7 activity than wild type (wt) cells. Moreover they exhibited an enhanced susceptibility for oxidative stress. Thus GPx2 protects the proliferative crypt base cells of the intestine, especially when the selenium supply is limited. Additionally an enhanced expression of tumor necrosis factor α and an enhanced infiltration of macrophages were detected in the colon of GPx2 KO-mice in comparison to the wt. These effects were observed on a selenium deficient (-Se) and -adequate (+Se) diet, but could be prevented by feeding a selenium supplemented (++Se) diet. Accordingly, GPx2 KO-mice have a basal low grade inflammation. This underlines, that GPx2 has an important anti-inflammatory function in the intestinal epithelium. Selenium deficiency is linked to an increased risk of developing colorectal cancer. In a mouse model of colitis-associated colon carcinogenesis, GPx2 had anti-inflammatory and thus anticarcinogenic effects. However, also procarcinogenic functions of the GPx2 have been observed. Therefore, this study aimed to analyse the role of GPx2 in a model of non-inflammation triggered, sporadic colon carcinogenesis induced by azoxymethane (AOM). In preneoplastic lesions of wt mice, an enhanced expression of GPx2 in comparison to the normal mucosa was frequently observed. An upregulation of GPx2 expression has also been described in human colon carcinogenesis. GPx2 KO mice had less tumors (-Se and ++Se) and less preneoplastic lesions (-Se, +Se) than wt mice. Accordingly GPx2 promotes colon carcinogenesis in the AOM-model. Eight hours after AOM-application, a higher rate of apoptosis was observed in the mid-crypt region of the colon of GPx2 ko mice in comparison to wt mice in the –Se and +Se groups, but not in the ++Se group or in the crypt base. Thus GPx2 might act procarcinogenic by preventing the elimination of cells with DNA-damage in the tumor initiation stage. Similarly, the enhanced GPx2-expression in preneoplastic cells could promote tumorigenesis by protecting these cells from oxidative stress, apoptosis or antitumor immunity. This effect might be enhanced by other selenoproteins like GPx1 or thioredoxin reductases that have also been reported to possess procarcinogenic properties and it might be closely related to the regulation of the redox state of tumor cells. In wt mice, the selenium content of the diet turned out to have a rather U-shaped effect on colon carcinogenesis. In the –Se and ++Se groups, wt mice tended to have more and larger tumors than in the ++Se group. In conclusion, GPx2 protects the proliferating cells of the intestinal crypt base, but it could also protect proliferating transformed cells and thus promote sporadic, AOM-induced colon carcinogenesis. In contrast, GPx2 acted anticarcinogenic in a model of colitis-associated colon carcinogenesis due to its antiinflammatory properties. Thus, the role of GPx2 in colon carcinogenesis depends on the underlying mechanisms, especially on the involvement of an inflammation.

Studies described in this thesis were aimed at characterizing the mechanism(s) of aflatoxin B1 (AFB1) pulmonary carcinogenesis by addressing the formation, prevention, and repair of AFB1-induced oxidative DNA damage. The ability of AFB1 to cause oxidative DNA damage in different lung cell types of the A/J mouse was examined. The formation of 8-hydroxy-2’-deoxyguanosine (8-OHdG) in freshly isolated mouse lung alveolar macrophages, alveolar type II cells, and nonciliated bronchial epithelial (Clara) cells, was assessed by high performance liquid chromatography with electrochemical detection. An increase in 8-OHdG formation occurred in macrophage and Clara cell preparations isolated from A/J mice two hours following in vivo treatment with a single tumourigenic dose of AFB1. Prior treatment with polyethylene glycol-conjugated catalase (PEG-CAT) prevented the AFB1-induced increase in 8-OHdG levels in all mouse lung cell preparations. These results support the possibility that oxidative DNA damage in mouse lung cells contributes to AFB1 carcinogenicity. Mouse lung tumourigenesis was assessed following treatment of A/J mice with PEG-CAT and/or AFB1. Unexpectedly, the mean number of tumours per mouse and tumour size in the PEG-CAT + AFB1 group were greater than those of the group treated with AFB1 alone. There was no difference in K-ras exon 1 mutation spectrum or in the histological diagnosis of tumours between treatment groups. In vitro incubation with mouse liver catalase (CAT) resulted in conversion of [3H]AFB1 into a DNA-binding species, a possible explanation for the results observed in vivo. These results demonstrate that PEG-CAT is not protective against AFB1 carcinogenicity in mouse lung despite preventing DNA oxidation. The effect of in vivo treatment of mice with AFB1 on pulmonary and hepatic base excision repair (BER) activities and levels of 8-oxoguanine DNA glycosylase (OGG1) was investigated. AFB1 treatment increased 8-OHdG levels and BER activity in mouse lung, but did not significantly affect either in liver. Levels of OGG1 immunoreactive protein were increased in both mouse lung and liver. These results indicate that oxidative DNA damage may be an important mechanism in the carcinogenicity of AFB1. However, BER activity is increased by AFB1 treatment, possibly representing a compensatory response to the production of oxidative DNA damage. / Thesis (Ph.D, Pharmacology & Toxicology) -- Queen's University, 2008-12-12 10:00:44.81