Polymorphic low penetrance genes and breast cancer : The role of genes involved in metabolism of xenobiotics, estrogens and reactive oxygen species

Various endogenous and exogenous factors have been reported to increase the risk of breast cancer. Many of those are related to prolonged lifetime exposure to estrogens. Furthermore, a positive family history of breast cancer and certain benign breast diseases are known to increase the risk of breast cancer. The role of lifestyle factors, such as use of alcohol and smoking has been an area of intensive study. Alcohol has been found to increase the risk of breast cancer, whereas the role of smoking has remained obscure. A multitude of enzymes are involved in the metabolism of estrogens and xenobiotics including the carcinogens found in tobacco smoke. Many of the metabolic enzymes exhibit genetic polymorphisms that can lead to inter-individual differences in their abilities to modify hazardous substrates. Therefore, in presence of a given chemical exposure, one subgroup of women may be more susceptible to breast carcinogenesis, since they carry unfavourable forms of the polymorphic genes involved in the metabolism of the chemical. In this work, polymorphic genes encoding for cytochrome P450 (CYP) 1A1 and 1B1, N-acetyl transferase 2 (NAT2), sulfotransferase 1A1 (SULT1A1), manganese superoxide dismutase (MnSOD) and vitamin D receptor (VDR) were investigated in relation to breast cancer susceptibility in a Finnish population. CYP1A1, CYP1B1 and SULT1A1 are involved in the metabolism of both estrogens and xenobiotics, whereas NAT2 is involved only in the latter. MnSOD is an antioxidant enzyme protecting cells from oxidative damage. VDR, in turn, mediates the effects of the active form of vitamin D (1,25(OH)2D3, calcitriol) on maintenance of calcium homeostasis and it has anti-proliferative effects in many cancer cells. A 1.3-fold (95% CIs 1.01-1.73) increased risk of breast cancer was seen among women who carried the NAT2 slow acetylator genotype and a 1.5-fold (95% CI 1.1-2.0) risk was found in women with a MnSOD variant A allele containing genotypes compared to women with the NAT2 rapid acetylator genotype or to those with the MnSOD VV genotype, respectively. Instead, women with the VDR a allele containing genotypes were found to be at a decreased risk for breast cancer (OR 0.73; 95% CI 0.54-0.98) compared to women with the AA genotype. No significant overall associations were found between SULT1A1 or CYP genotypes and breast cancer risk, whereas a combination of the CYP1B1 432Val allele containing genotypes with the NAT2 slow acetylator genotypes posed a 1.5-fold (95% CI 1.03-2.24) increased risk. Moreover, NAT2 slow acetylator genotype was found to be confined to women with an advanced stage of breast cancer (stages III and IV). Further evidence for the association of xenobiotic metabolising genes with breast cancer risk was found when active smoking was taken into account. Women who smoked less than 10 cigarettes/day and carried at least one CYP1B1 432Val variant allele, were at 3.1-fold (95% CI 1.32-7.12) risk of breast cancer compared to women who smoked the same amount but did not carry the variant allele. Furthermore, the risk was significantly increased with increasing number of the CYP1B1 432Val alleles (p for trend 0.005). In addition, women who smoked less than 5 pack-years and carried the NAT2 slow acetylator genotype were at a 2.6-fold (95% CI 1.01-6.48) increased risk of breast cancer compared to women who smoked the same amount but carried the NAT2 rapid acetylator genotype. Furthermore, the combination of the CYP1B1 432Val allele and the NAT2 slow acetylator genotype increased the risk of breast cancer by 2.5-fold (95% CI 1.11-5.45) among ever smokers. Instead, the MnSOD A allele was found to be a risk factor among postmenopausal long-term smokers (>15 years of smoking) (OR 5.1; 95% CI 1.4-18.4) or among postmenopausal women who had smoked more than 10 cigarettes/day (OR 5.5; 95% CI 1.3-23.4) compared to women who had similar smoking habits but carried the MnSOD V/V genotype. Similarly, within subgroups of postmenopausal women who were using oral contraceptives, hormone replacement therapy or alcohol, women carrying the MnSOD A allele genotypes seemed to be at increased risk of breast cancer compared to women with the MnSOD V/V genotype. A positive family history of breast cancer and high parity were shown to be inversely associated with breast cancer risk among women carrying the VDR ApaI a allele or among premenopausal women carrying the SULT1A1*2 allele, respectively.

Postmenopausal hot flushes, vascular health and hormone therapy

Vasomotor hot flushes are complained of by approximately 75% of postmenopausal women, but their frequency and severity show great individual variation. Hot flushes have been present in women attending observational studies showing cardiovascular benefit associated with hormone therapy use, whereas they have been absent or very mild in randomized hormone therapy trials showing cardiovascular harm. Therefore, if hot flushes are a factor connected with vascular health, they could perhaps be one explanation for the divergence of cardiovascular data in observational versus randomized studies. For the present study 150 healthy, recently postmenopausal women showing a large variation in hot flushes were studied in regard to cardiovascular health by way of pulse wave analysis, ambulatory blood pressure and several biochemical vascular markers. In addition, the possible impact of hot flushes on outcomes of hormone therapy was studied. This study shows that women with severe hot flushes exhibit a greater vasodilatory reactivity as assessed by pulse wave analysis than do women without vasomotor symptoms. This can be seen as a hot flush-related vascular benefit. Although severe night-time hot flushes seem to be accompanied by transient increases in blood pressure and heart rate, the diurnal blood pressure and heart rate profiles show no significant differences between women without and with mild, moderate or severe hot flushes. The levels of vascular markers, such as lipids, lipoproteins, C-reactive protein and sex hormone-binding globulin show no association with hot flush status. In the 6-month hormone therapy trial the women were classified as having either tolerable or intolerable hot flushes. These groups were treated in a randomized order with transdermal estradiol gel, oral estradiol alone or in combination with medroxyprogesterone acetate, or with placebo. In women with only tolerable hot flushes, oral estradiol leads to a reduced vasodilatory response and increases in 24-hour and daytime blood pressures as compared to women with intolerable hot flushes receiving the same therapy. No such effects were observed with the other treatment regimes or in women with intolerable hot flushes. The responses of vascular biomarkers to hormone therapy are unaffected by hot flush status. In conclusion, hot flush status contributes to cardiovascular health before and during hormone therapy. Severe hot flushes are associated with an increased vasodilatory, and thus, a beneficial vascular status. Oral estradiol leads to vasoconstrictive changes and increases in blood pressure, and thus to possible vascular harm, but only in women whose hot flushes are so mild that they would probably not lead to the initiation of hormone therapy in clinical practice. Healthy, recently postmenopausal women with moderate to severe hot flushes should be given the opportunity to use hormone therapy alleviate hot flushes, and if estrogen is prescribed for indications other than for the control of hot flushes, transdermal route of administration should be favored.

A Nationwide Study on Breast Cancer Risk in Postmenopausal Women Using Hormone Therapy in Finland

Since national differences exist in genes, environment, diet and life habits and also in the use of postmenopausal hormone therapy (HT), the associations between different hormone therapies and the risk for breast cancer were studied among Finnish postmenopausal women. All Finnish women over 50 years of age who used HT were identified from the national medical reimbursement register, established in 1994, and followed up for breast cancer incidence (n= 8,382 cases) until 2005 with the aid of the Finnish Cancer Registry. The risk for breast cancer in HT users was compared to that in the general female population of the same age. Among women using oral or transdermal estradiol alone (ET) (n = 110,984) during the study period 1994-2002 the standardized incidence ratio (SIR) for breast cancer in users for < 5 years was 0.93 (95% confidence interval (CI) 0.80–1.04), and in users for ≥ 5 years 1.44 (1.29–1.59). This therapy was associated with similar rises in ductal and lobular types of breast cancer. Both localized stage (1.45; 1.26–1.66) and cancers spread to regional nodes (1.35; 1.09–1.65) were associated with the use of systemic ET. Oral estriol or vaginal estrogens were not accompanied with a risk for breast cancer. The use of estrogen-progestagen therapy (EPT) in the study period 1994-2005 (n= 221,551) was accompanied with an increased incidence of breast cancer (1.31;1.20-1.42) among women using oral or transdermal EPT for 3-5 years, and the incidence increased along with the increasing duration of exposure (≥10 years, 2.07;1.84-2.30). Continuous EPT entailed a significantly higher (2.44; 2.17-2.72) breast cancer incidence compared to sequential EPT (1.78; 1.64-1.90) after 5 years of use. The use of norethisterone acetate (NETA) as a supplement to estradiol was accompanied with a higher incidence of breast cancer after 5 years of use (2.03; 1.88-2.18) than that of medroxyprogesterone acetate (MPA) (1.64; 1.49-1.79). The SIR for the lobular type of breast cancer was increased within 3 years of EPT exposure (1.35; 1.18-1.53), and the incidence of the lobular type of breast cancer (2.93; 2.33-3.64) was significantly higher than that of the ductal type (1.92; 1.67-2.18) after 10 years of exposure. To control for some confounding factors, two case control studies were performed. All Finnish women between the ages of 50-62 in 1995-2007 and diagnosed with a first invasive breast cancer (n= 9,956) were identified from the Finnish Cancer Registry, and 3 controls of similar age (n=29,868) without breast cancer were retrieved from the Finnish national population registry. Subjects were linked to the medical reimbursement register for defining the HT use. The use of ET was not associated with an increased risk for breast cancer (1.00; 0.92-1.08). Neither was progestagen-only therapy used less than 3 years. However, the use of tibolone was associated with an elevated risk for breast cancer (1.39; 1.07-1.81). The case-control study confirmed the results of EPT regarding sequential vs. continuous use of progestagen, including progestagen released continuously by an intrauterine device; the increased risk was seen already within 3 years of use (1.65;1.32-2.07). The dose of NETA was not a determinant as regards the breast cancer risk. Both systemic ET, and EPT are associated with an elevation in the risk for breast cancer. These risks resemble to a large extent those seen in several other countries. The use of an intrauterine system alone or as a complement to systemic estradiol is also associated with a breast cancer risk. These data emphasize the need for detailed information to women who are considering starting the use of HT.

Postoperative Volume Therapy in Cardiac Surgery : Effects on Hemostatic and Circulatory Variables

Background: Patients may need massive volume-replacement therapy after cardiac surgery because of large fluid transfer perioperatively, and the use of cardiopulmonary bypass. Hemodynamic stability is better maintained with colloids than crystalloids but colloids have more adverse effects such as coagulation disturbances and impairment of renal function than do crystalloids. The present study examined the effects of modern hydroxyethyl starch (HES) and gelatin solutions on blood coagulation and hemodynamics. The mechanism by which colloids disturb blood coagulation was investigated by thromboelastometry (TEM) after cardiac surgery and in vitro by use of experimental hemodilution. Materials and methods: Ninety patients scheduled for elective primary cardiac surgery (Studies I, II, IV, V), and twelve healthy volunteers (Study III) were included in this study. After admission to the cardiac surgical intensive care unit (ICU), patients were randomized to receive different doses of HES 130/0.4, HES 200/0.5, or 4% albumin solutions. Ringer’s acetate or albumin solutions served as controls. Coagulation was assessed by TEM, and hemodynamic measurements were based on thermodilutionally measured cardiac index (CI). Results: HES and gelatin solutions impaired whole blood coagulation similarly as measured by TEM even at a small dose of 7 mL/kg. These solutions reduced clot strength and prolonged clot formation time. These effects were more pronounced with increasing doses of colloids. Neither albumin nor Ringer’s acetate solution disturbed blood coagulation significantly. Coagulation disturbances after infusion of HES or gelatin solutions were clinically slight, and postoperative blood loss was comparable with that of Ringer’s acetate or albumin solutions. Both single and multiple doses of all the colloids increased CI postoperatively, and this effect was dose-dependent. Ringer’s acetate had no effect on CI. At a small dose (7 mL/kg), the effect of gelatin on CI was comparable with that of Ringer’s acetate and significantly less than that of HES 130/0.4 (Study V). However, when the dose was increased to 14 and 21 mL/kg, the hemodynamic effect of gelatin rose and became comparable with that of HES 130/0.4. Conclusions: After cardiac surgery, HES and gelatin solutions impaired clot strength in a dose-dependent manner. The potential mechanisms were interaction with fibrinogen and fibrin formation, resulting in decreased clot strength, and hemodilution. Although the use of HES and gelatin inhibited coagulation, postoperative bleeding on the first postoperative morning in all the study groups was similar. A single dose of HES solutions improved CI postoperatively more than did gelatin, albumin, or Ringer’s acetate. However, when administered in a repeated fashion, (cumulative dose of 14 mL/kg or more), no differences were evident between HES 130/0.4 and gelatin.

Mechanisms and molecular regulation of mammalian tooth replacement

In most non-mammalian vertebrates, such as fish and reptiles, teeth are replaced continuously. However, tooth replacement in most mammals, including human, takes place only once and further renewal is apparently inhibited. It is not known how tooth replacement is genetically regulated, and little is known on the physiological mechanism and evolutionary reduction of tooth replacement in mammals. In this study I have attempted to address these questions. In a rare human condition cleidocranial dysplasia, caused by a mutation in a Runt domain transcription factor Runx2, tooth replacement is continued. Runx2 mutant mice were used to investigate the molecular mechanisms of Runx2 function. Microarray analysis from dissected embryonic day 14 Runx2 mutant and wild type dental mesenchymes revealed many downstream targets of Runx2, which were validated using in situ hybridization and tissue culture methods. Wnt signaling inhibitor Dkk1 was identified as a candidate target, and in tissue culture conditions it was shown that Dkk1 is induced by FGF4 and this induction is Runx2 dependent. These experiments demonstrated a connection between Runx2, FGF and Wnt signaling in tooth development and possibly also in tooth replacement. The role of Wnt signaling in tooth replacement was further investigated by using a transgenic mouse model where Wnt signaling mediator β-catenin is continuously stabilized in dental epithelium. This stabilization led to activated Wnt signaling and to the formation of multiple enamel knots. In vitro and transplantation experiments were performed to examine the process of extra tooth formation. We showed that new teeth were continuously generated and that new teeth form from pre-existing teeth. A morphodynamic activator-inhibitor model was used to simulate enamel knot formation. By increasing the intrinsic production rate of the activator (β-catenin), the multiple enamel knot phenotype was reproduced by computer simulations. It was thus concluded that β-catenin acts as an upstream activator of enamel knots, closely linking Wnt signaling to the regulation of tooth renewal. As mice do not normally replace teeth, we used other model animals to investigate the physiological and genetic mechanisms of tooth replacement. Sorex araneus, the common shrew was earlier reported to have non-functional tooth replacement in all antemolar tooth positions. We showed by histological and gene expression studies that there is tooth replacement only in one position, the premolar 4 and that the deciduous tooth is diminished in size and disappears during embryogenesis without becoming functional. The growth rates of deciduous and permanent premolar 4 were measured and it was shown by competence inference that the early initiation of the replacement tooth in relation to the developmental stage of the deciduous tooth led to the inhibition of deciduous tooth morphogenesis. It was concluded that the evolutionary loss of deciduous teeth may involve the early activation of replacement teeth, which in turn suppress their predecessors. Mustela putorius furo, the ferret, has a dentition that resembles that of the human as ferrets have teeth that belong to all four tooth families, and all the antemolar teeth are replaced once. To investigate the replacement mechanism, histological serial sections from different embryonic stages were analyzed. It was noticed that tooth replacement is a process which involves the growth and detachment of the dental lamina from the lingual cervical loop of the deciduous tooth. Detachment of the deciduous tooth leads to a free successional dental lamina, which grows deeper into the mesenchyme, and later buds the replacement tooth. A careful 3D analysis of serial histological sections was performed and it was shown that replacement teeth are initiated from the successional dental lamina and not from the epithelium of the deciduous tooth. The molecular regulation of tooth replacement was studied and it was shown by examination of expression patterns of candidate regulatory genes that BMP/Wnt inhibitor Sostdc1 was strongly expressed in the buccal aspect of the dental lamina, and in the intersection between the detaching deciduous tooth and the successional dental lamina, suggesting a role for Sostdc1 in the process of detachment. Shh was expressed in the enamel knot and in the inner enamel epithelium in both generations of teeth supporting the view that the morphogenesis of both generations of teeth is regulated by similar mechanisms. In summary, histological and molecular studies on different model animals and transgenic mouse models were used to investigate tooth replacement. This thesis work has significantly contributed to the knowledge on the physiological mechanisms and molecular regulation of tooth replacement and its evolutionary suppression in mammals.

Inpatient hospital care and its costs among type 1 diabetic patients in Finland : a nationwide longitudinal study

Background. In Finland, the incidence of type 1 diabetes mellitus (T1DM) is the highest in the world, and it continues to increase steadily. No effective preventative interventions exist either for individuals at high risk or for the population as a whole. In addition to problems with daily lifelong insulin replacement therapy, T1DM patients with long-lasting disease suffer from various diabetes related complications. The complications can lead to severe impairments and reductions in functional capacity and quality of life and in the worst case they can be fatal. Longitudinal studies on the costs of T1DM are extremely rare, especially in Finland. Typically, in these studies, distinctions between the various types of diabetes have not been made, and costs have not been calculated separately for the sexes. Aims. The aim of this study was to describe inpatient hospital care and costs of inpatient care in a cohort of 5,166 T1DM patients by sex during 1973-1998 in Finland. Inpatient care and costs of care due to T1DM without complications, due to T1DM with complications and due to other causes were calculated separately. Material and Methods. The study population consisted of all Finnish T1DM patients diagnosed before the age of 18 years between January 1st in 1965 and December 31st in 1979 and derived from the Finnish population based T1DM register (N=5,120 in 1979 and N=4,701 in 1997). Data on hospitalisations were obtained from the Finnish Hospital Discharge Register. Results. In the early stages of T1DM, the majority of the use of inpatient care was due to the treatment of T1DM without complications. There were enormous increases in the use of inpatient care for certain complications when T1DM lasted longer (from 9.5 years to 16.5 years). For women, the yearly number of bed-days for renal complications increased 4.8-fold, for peripheral vascular disease 4.3-fold and for ophthalmic complications 2.5-fold. For men, the corresponding increases were as follows: 5-fold, 6.9-fold and 2.5-fold. The yearly bed-days for glaucoma increased 8-fold, nephropathy 7-fold and microangiopathy 6-fold in the total population. During these 7 years, the yearly numbers of bed-days for T1DM without complications dropped dramatically. The length of stay in inpatient care decreased notably, but hospital visits became more frequent when the length of duration of T1DM increased from 9.5 years to 16.5 years. The costs of treatments due to complications increased when T1DM lasted longer. Costs due to inpatient care of complications in the cohort 2.5-folded as duration of T1DM increased from 9.5 years to 16.5 years, while the total costs of inpatient care in the cohort dropped by 22% due to an 80% decrease in the costs of care of T1DM without complications. Treating complications of female patients was more expensive than treating complications of men when T1DM had lasted 9.5 years; the mean annual costs for inpatient care of a female diabetic (any cause) were 1,642 , and the yearly costs of care of complications were 237 . The corresponding yearly mean costs for a male patient were 1,198 and 167 . Treating complications of female patients was more expensive than that of male patients also when the duration of diabetes was 16.5 years, although the difference in average annual costs between sexes was somewhat smaller. Conclusions. In the early phases of T1DM, the treatment of T1DM without complications causes a considerable amount of hospital bed-days. The use of inpatient care due to complications of T1DM strongly increases with ageing of patients. The economic burden of inpatient care of T1DM is substantial.

IgA nephropathy and Henoch-Schönlein nephritis in adults : with special reference to factors affecting outcome

IgA nephropathy (IgAN) is the most common primary glomerulonephritis. In one third of the patients the disease progresses, and they eventually need renal replacement therapy. IgAN is in most cases a slowly progressing disease, and the prediction of progression has been difficult, and the results of studies have been conflicting. Henoch-Schönlein nephritis (HSN) is rare in adults, and prediction of the outcome is even more difficult than in IgAN. This study was conducted to evaluate the clinical and histopathological features and predictors of the outcome of IgAN and HSN diagnosed in one centre (313 IgAN patients and 38 HSN patients), and especially in patients with normal renal function at the time of renal biopsy. The study also aimed to evaluate whether there is a difference in the progression rates in four countries (259 patients from Finland, 112 from UK, 121 from Australia and 274 from Canada), and if so, can this be explained by differences in renal biopsy policy. The third aim was to measure urinary excretions of cytokines interleukin 1ß (IL-1ß) and interleukin 1 receptor antagonist (IL-1ra) in patients with IgAN and HSN and the correlations of excretion of these substances with histopathological damage and clinical factors. A large proportion of the patients diagnosed in Helsinki as having IgAN had normal renal function (161/313 patients). Four factors, (hypertension, higher amounts of urinary erythrocytes, severe arteriolosclerosis and a higher glomerular score) which independently predicted progression (logistic regression analysis), were identified in mild disease. There was geographic variability in renal survival in patients with IgAN. When age, levels of renal function, proteinuria and blood pressure were taken into account, it showed that the variability related mostly to lead-time bias and renal biopsy indications. Amount of proteinuria more than 0.4g/24h was the only factor that was significantly related to the progression of HSN. the Hypertension and the level of renal function were found to be factors predicting outcome in patients with normal renal function at the time of diagnosis. In IgAN patients, IL-1ra excretion into urine was found to be decreased as compared with HSN patients and healthy controls. Patients with a high IL-1ra/IL-1ß ratio had milder histopathological changes in renal biopsy than patients with a low/normal IL-1ra/IL-1ß ratio. It was also found that the excretion of IL-1ß and especially IL-1ra were significantly higher in women. In conclusion, it was shown that factors associated with outcome can reliably be identified even in mild cases of IgAN. Predicting outcome in adult HSN, however, remains difficult.

Hormone therapy in elderly women; a comparative study with alendronate of the effects on bone, cardiovascular risk factors, periodontal conditions and quality of life

Thirty percent of 70-year-old women have osteoporosis; after age of 80 its prevalence is up to 70%. Postmenopausal women with osteoporosis seem to be at an increased risk for cardiovascular events, and deterioration of oral health, as shown by attachment loss of teeth, which is proportional to the severity of osteoporosis. Osteoporosis can be treated with many different medication, e.g. estrogen and alendronate. We randomized 90 elderly osteoporotic women (65-80 years of age) to receive hormone therapy (HT)(2mg E2+NETA), 10mg alendronate, and their combination for two years and compared their effects on bone mineral density (BMD) and turnover, two surrogate markers of the risk of cardiovascular diseases, C-reactive protein (CRP) and E-selectin, as well as oral health. The effect of HT on health-related quality of life (HRQoL) was studied in the population-based cohort of 1663 postmenopausal women (mean age 68 yr) (585 estrogen users and 1078 non-users). BMD was measured with dual-energy X-ray absorptiometry (DXA) at 0, 12 and 24 months. Urinary N-telopeptide (NTX) of type I collagen, a marker of bone resorption, and serum aminoterminal propeptide of human type I procollagen (PINP), a marker of bone formation, were measured every six months of treatment. Serum CRP and E-selectin, were measured at 0, 6, and 12 months. Dental, and periodontal conditions, and gingival crevicular fluid (GCF) matrix metalloproteinase (MMP)-8 levels were studied to evaluate the oral health status and for the mouth symptoms a structured questionnaire was used. The HRQoL was measured with 15D questionnaire. Lumbar spine BMD increased similarly in all treatment groups (6.8-8.4% and 9.1-11.2%). Only HT increased femoral neck BMD at both 12 (4.9%) and 24 months (5.8%), at the latter time point the HT group differed significantly from the other groups. HT reduced bone marker levels of NTX and PINP significantly less than other two groups.Oral HT significantly increased serum CRP level by 76.5% at 6 and by 47.1% (NS) at 12 months, and decreased serum E-selectin level by 24.3% and 30.0%. Alendronate had no effect on these surrogate markers. Alendronate caused a decrease in the resting salivary flow rate and tended to increase GCF MMP-8 levels. Otherwise, there was no effect on the parameters of oral health. HT improved the HRQoL of elderly women significantly on the dimensions of usual activities, vitality and sexual activity, but the overall improvement in HRQoL was neither statistically significant nor clinically important. In conclusion, bisphosphonates might be the first option to start the treatment of postmenopausal osteoporosis in the old age.

Part I: Enzyme replacement versus neurotrophic factor viral vector-mediated gene therapy of Parkinson’s disease, Part II: The effects of orally dosed tolcapone and entacapone on dopamine metabolism in the dorsal striatum and nucleus accumbens in rats

Part I: Parkinson’s disease is a slowly progressive neurodegenerative disorder in which particularly the dopaminergic neurons of the substantia nigra pars compacta degenerate and die. Current conventional treatment is based on restraining symptoms but it has no effect on the progression of the disease. Gene therapy research has focused on the possibility of restoring the lost brain function by at least two means: substitution of critical enzymes needed for the synthesis of dopamine and slowing down the progression of the disease by supporting the functions of the remaining nigral dopaminergic neurons by neurotrophic factors. The striatal levels of enzymes such as tyrosine hydroxylase, dopadecarboxylase and GTP-CH1 are decreased as the disease progresses. By replacing one or all of the enzymes, dopamine levels in the striatum may be restored to normal and behavioral impairments caused by the disease may be ameliorated especially in the later stages of the disease. The neurotrophic factors glial cell derived neurotrophic factor (GDNF) and neurturin have shown to protect and restore functions of dopaminergic cell somas and terminals as well as improve behavior in animal lesion models. This therapy may be best suited at the early stages of the disease when there are more dopaminergic neurons for neurotrophic factors to reach. Viral vector-mediated gene transfer provides a tool to deliver proteins with complex structures into specific brain locations and provides long-term protein over-expression. Part II: The aim of our study was to investigate the effects of two orally dosed COMT inhibitors entacapone (10 and 30 mg/kg) and tolcapone (10 and 30 mg/kg) with a subsequent administration of a peripheral dopadecarboxylase inhibitor carbidopa (30 mg/kg) and L- dopa (30 mg/kg) on dopamine and its metabolite levels in the dorsal striatum and nucleus accumbens of freely moving rats using dual-probe in vivo microdialysis. Earlier similarly designed studies have only been conducted in the dorsal striatum. We also confirmed the result of earlier ex vivo studies regarding the effects of intraperitoneally dosed tolcapone (30 mg/kg) and entacapone (30 mg/kg) on striatal and hepatic COMT activity. The results obtained from the dorsal striatum were generally in line with earlier studies, where tolcapone tended to increase dopamine and DOPAC levels and decrease HVA levels. Entacapone tended to keep striatal dopamine and HVA levels elevated longer than in controls and also tended to elevate the levels of DOPAC. Surprisingly in the nucleus accumbens, dopamine levels after either dose of entacapone or tolcapone were not elevated. Accumbal DOPAC levels, especially in the tolcapone 30 mg/kg group, were elevated nearly to the same extent as measured in the dorsal striatum. Entacapone 10 mg/kg elevated accumbal HVA levels more than the dose of 30 mg/kg and the effect was more pronounced in the nucleus accumbens than in the dorsal striatum. This suggests that entacapone 30 mg/kg has minor central effects. Also our ex vivo study results obtained from the dorsal striatum suggest that entacapone 30 mg/kg has minor and transient central effects, even though central HVA levels were not suppressed below those of the control group in either brain area in the microdialysis study. Both entacapone and tolcapone suppressed hepatic COMT activity more than striatal COMT activity. Tolcapone was more effective than entacapone in the dorsal striatum. The differences between dopamine and its metabolite levels in the dorsal striatum and nucleus accumbens may be due to different properties of the two brain areas.

Molecular and Clinical Characteristics of Pituitary Adenoma Predisposition (PAP)

Pituitary adenomas are common benign neoplasms. Although most of them are sporadic, a minority occurs in familial settings. Heterozygous germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene were found to underlie familial pituitary adenomas, a condition designated as pituitary adenoma predisposition (PAP). PAP confers incomplete penetrance of mostly growth hormone (GH) secreting adenomas in young patients, who often lack a family history of pituitary adenomas. This thesis work aimed to clarify the molecular and clinical characteristics of PAP. Applying the multiplex ligation-dependent probe amplification assay (MLPA), we found large genomic AIP deletions to account for a subset of PAP. Therefore, MLPA could be considered in PAP suspected patients with no AIP mutations found with conventional sequencing. We generated an Aip mouse model to examine pituitary tumorigenesis in vivo. The heterozygous Aip mutation conferred complete penetrance of pituitary adenomas that were mostly GH-secreting, rendering the phenotype of the Aip mouse similar to that of PAP patients. We suggest that AIP may function as a candidate gatekeeper gene in somatotrophs. To clarify molecular mechanisms of tumorigenesis, we elucidated the expression of AIP-related molecules in human and mouse pituitary tumors. The expression of aryl hydrocarbon receptor nuclear translocator (ARNT) was reduced in mouse Aip-deficient adenomas, and similar ARNT reduction was also evident in human AIP mutation positive adenomas. This suggests that in addition to participating in the hypoxia pathway, estrogen receptor signaling and xenobiotic response pathways, ARNT may play a role in AIP-related tumorigenesis. We also studied the characteristics and the response to therapy of PAP patients and found them to have an aggressive disease phenotype with young age at onset. Therefore, improvement in treatment outcomes of PAP patients would require their efficient identification and earlier diagnosis of the pituitary adenomas. The possible role of the RET proto-oncogene in tumorigenesis of familial AIP mutation negative pituitary adenomas was evaluated, but none of the found RET germline variants were considered pathogenic. Surprisingly, RET immunohistochemistry suggested possible underexpression of RET in AIP mutation positive pituitary adenomas an observation that merits further investigation.

Molecular Mechanisms of Androgen Receptor Interactions

The androgen receptor (AR) mediates the effects of the male sex-steroid hormones (androgens), testosterone and 5?-dihydrotestosterone. Androgens are critical in the development and maintenance of male sexual characteristics. AR is a member of the steroid receptor ligand-inducible transcription factor family. The steroid receptor family is a subgroup of the nuclear receptor superfamily that also includes receptors for the active forms of vitamin A, vitamin D3, and thyroid hormones. Like all nuclear receptors, AR has a conserved modular structure consisting of a non-conserved amino-terminal domain (NTD), containing the intrinsic activation function 1, a highly conserved DNA-binding domain, and a conserved ligand-binding domain (LBD) that harbors the activation function 2. Each of these domains plays an important role in receptor function and signaling, either via intra- and inter-receptor interactions, interactions with specific DNA sequences, termed hormone response elements, or via functional interactions with domain-specific proteins, termed coregulators (coactivators and corepressors). Upon binding androgens, AR acquires a new conformational state, translocates to the nucleus, binds to androgen response elements, homodimerizes and recruits sequence-specific coregulatory factors and the basal transcription machinery. This set of events is required to activate gene transcription (expression). Gene transcription is a strictly modulated process that governs cell growth, cell homeostasis, cell function and cell death. Disruptions of AR transcriptional activity caused by receptor mutations and/or altered coregulator interactions are linked to a wide spectrum of androgen insensitivity syndromes, and to the pathogenesis of prostate cancer (CaP). The treatment of CaP usually involves androgen depletion therapy (ADT). ADT achieves significant clinical responses during the early stages of the disease. However, under the selective pressure of androgen withdrawal, androgen-dependent CaP can progress to an androgen-independent CaP. Androgen-independent CaP is invariably a more aggressive and untreatable form of the disease. Advancing our understanding of the molecular mechanisms behind the switch in androgen-dependency would improve our success of treating CaP and other AR related illnesses. This study evaluates how clinically identified AR mutations affect the receptor s transcriptional activity. We reveal that a potential molecular abnormality in androgen insensitivity syndrome and CaP patients is caused by disruptions of the important intra-receptor NTD/LBD interaction. We demonstrate that the same AR LBD mutations can also disrupt the recruitment of the p160 coactivator protein GRIP1. Our investigations reveal that 30% of patients with advanced, untreated local CaP have somatic mutations that may lead to increases in AR activity. We report that somatic mutations that activate AR may lead to early relapse in ADT. Our results demonstrate that the types of ADT a CaP patient receives may cause a clustering of mutations to a particular region of the receptor. Furthermore, the mutations that arise before and during ADT do not always result in a receptor that is more active, indicating that coregulator interactions play a pivotal role in the progression of androgen-independent CaP. To improve CaP therapy, it is necessary to identify critical coregulators of AR. We screened a HeLa cell cDNA library and identified small carboxyl-terminal domain phosphatase 2 (SCP2). SCP2 is a protein phosphatase that directly interacts with the AR NTD and represses AR activity. We demonstrated that reducing the endogenous cellular levels of SCP2 causes more AR to load on to the prostate specific antigen (PSA) gene promoter and enhancer regions. Additionally, under the same conditions, more RNA polymerase II was recruited to the PSA promoter region and overall there was an increase in androgen-dependent transcription of the PSA gene, revealing that SCP2 could play a role in the pathogenesis of CaP.

Molecular Modelling of Estrogen-Producing Enzymes CYP450 Aromatase and 17beta-Hydroxysteroid Dehydrogenase Type 1

Breast cancer is the most common cancer in women in the western countries. Approximately two-thirds of breast cancer tumours are hormone dependent, requiring estrogens to grow. Estrogens are formed in the human body via a multistep route starting from cholesterol. The final steps in the biosynthesis include the CYP450 aromatase enzyme, converting the male hormones androgens (preferred substrate androstenedione ASD) into estrogens(estrone E1), and the 17beta-HSD1 enzyme, converting the biologically less active E1 into the active hormone 17beta-hydroxyestradiol E2. E2 is bound to the nuclear estrogen receptors causing a cascade of biochemical reactions leading to cell proliferation in normal tissue, and to tumour growth in cancer tissue. Aromatase and 17beta-HSD1 are expressed in or near the breast tumour, locally providing the tissue with estrogens. One approach in treating hormone dependent breast tumours is to block the local estrogen production by inhibiting these two enzymes. Aromatase inhibitors are already on the market in treating breast cancer, despite the lack of an experimentally solved structure. The structure of 17beta-HSD1, on the other hand, has been solved, but no commercial drugs have emerged from the drug discovery projects reported in the literature. Computer-assisted molecular modelling is an invaluable tool in modern drug design projects. Modelling techniques can be used to generate a model of the target protein and to design novel inhibitors for them even if the target protein structure is unknown. Molecular modelling has applications in predicting the activities of theoretical inhibitors and in finding possible active inhibitors from a compound database. Inhibitor binding at atomic level can also be studied with molecular modelling. To clarify the interactions between the aromatase enzyme and its substrate and inhibitors, we generated a homology model based on a mammalian CYP450 enzyme, rabbit progesterone 21-hydroxylase CYP2C5. The model was carefully validated using molecular dynamics simulations (MDS) with and without the natural substrate ASD. Binding orientation of the inhibitors was based on the hypothesis that the inhibitors coordinate to the heme iron, and were studied using MDS. The inhibitors were dietary phytoestrogens, which have been shown to reduce the risk for breast cancer. To further validate the model, the interactions of a commercial breast cancer drug were studied with MDS and ligand–protein docking. In the case of 17beta-HSD1, a 3D QSAR model was generated on the basis of MDS of an enzyme complex with active inhibitor and ligand–protein docking, employing a compound library synthesised in our laboratory. Furthermore, four pharmacophore hypotheses with and without a bound substrate or an inhibitor were developed and used in screening a commercial database of drug-like compounds. The homology model of aromatase showed stable behaviour in MDS and was capable of explaining most of the results from mutagenesis studies. We were able to identify the active site residues contributing to the inhibitor binding, and explain differences in coordination geometry corresponding to the inhibitory activity. Interactions between the inhibitors and aromatase were in agreement with the mutagenesis studies reported for aromatase. Simulations of 17beta-HSD1 with inhibitors revealed an inhibitor binding mode with hydrogen bond interactions previously not reported, and a hydrophobic pocket capable of accommodating a bulky side chain. Pharmacophore hypothesis generation, followed by virtual screening, was able to identify several compounds that can be used in lead compound generation. The visualisation of the interaction fields from the QSAR model and the pharmacophores provided us with novel ideas for inhibitor development in our drug discovery project.

Ozone-induced signaling in Arabidopsis thaliana

Tropospheric ozone (O3) is one of the most common air pollutants in industrialized countries, and an increasing problem in rapidly industrialising and developing countries in Asia, Africa and South America. Elevated concentrations of tropospheric O3 can lead to decrease in photosynthesis rate and therefore affect the normal metabolism, growth and seed production. Acute and high O3 episodes can lead to extensive damage leading to dead tissue in plants. Thus, O3 derived growth defects can lead to reduction in crop yield thereby leading to economical losses. Despite the extensive research on this area, many questions remain open on how these processes are controlled. In this study, the stress-induced signaling routes and the components involved were elucidated in more detail starting from visual damage to changes in gene expression, signaling routes and plant hormone interactions that are involved in O3-induced cell death. In order to elucidate O3-induced responses in Arabidopsis, mitogen-activated protein kinase (MAPK) signaling was studied using different hormonal signaling mutants. MAPKs were activated at the beginning of the O3 exposure. The activity of MAPKs, which were identified as AtMPK3 and AtMPK6, reached the maximum at 1 and 2 hours after the start of the exposure, respectively. The activity decreased back to clean air levels at 8 hours after the start of the exposure. Both AtMPK3 and AtMPK6 were translocated to nucleus at the beginning of the O3 exposure where they most likely affect gene expression. Differences were seen between different hormonal signaling mutants. Functional SA signaling was shown to be needed for the full protein levels and activation of AtMPK3. In addition, AtMPK3 and AtMPK6 activation was not dependent on ethylene signaling. Finally, jasmonic acid was also shown to have an impact on AtMPK3 protein levels and AtMPK3 activity. To further study O3-induced cell death, an earlier isolated O3 sensitive Arabidopsis mutant rcd1 was mapped, cloned and further characterized. RCD1 was shown to encode a gene with WWE and ADP-ribosylation domains known to be involved in protein-protein interactions and cell signaling. rcd1 was shown to be involved in many processes including hormonal signaling and regulation of stress-responsive genes. rcd1 is sensitive against O3 and apoplastic superoxide, but tolerant against paraquat that produces superoxide in chloroplast. rcd1 is also partially insensitive to glucose and has alterations in hormone responses. These alterations are seen as ABA insensitivity, reduced jasmonic acid sensitivity and reduced ethylene sensitivity. All these features suggest that RCD1 acts as an integrative node in hormonal signaling and it is involved in the hormonal regulation of several specific stress-responsive genes. Further studies with the rcd1 mutant showed that it exhibits the classical features of programmed cell death, PCD, in response to O3. These include nuclear shrinkage, chromatin condensation, nuclear DNA degradation, cytosol vesiculation and accumulation of phenolic compounds and eventually patches of HR-like lesions. rcd1 was found to produce extensive amount of salicylic acid and jasmonic acid in response to O3. Double mutant studies showed that SA independent and dependent processes were involved in the O3-induced PCD in rcd1 and that increased sensitivity against JA led to increased sensitivity against O3. Furthermore, rcd1 had alterations in MAPK signature that resembled changes that were previously seen in mutants defective in SA and JA signaling. Nitric oxide accumulation and its impact on O3-induced cell death were also studied. Transient accumulation of NO was seen at the beginning of the O3 exposure, and during late time points, NO accumulation coincided with the HR-like lesions. NO was shown to modify defense gene expression, such as, SA and ethylene biosynthetic genes. Furthermore, rcd1 was shown to produce more NO in control conditions. In conclusion, NO was shown to be involved in O3-induced signaling leading to attenuation of SA biosynthesis and other defense related genes.