Reactions of Chlorambucil and its main metabolite, Phenylacetic Acid Mustard, with 2’-deoxyribonucleosides and Calf Thymus DNA

Chlorambucil is an anticancer agent used in the treatment of a variety of cancers, especially in chronic lymphocytic leukemia, and autoimmune diseases. Nevertheless, chlorambucil is potentially mutagenic, teratogenic and carcinogenic. The high antitumor activity and high toxicity of chlorambucil and its main metabolite, phenylacetic acid mustard, to normal tissues have been known for a long time. Despite this, no detailed chemical data on their reactions with biomolecules in aqueous media have been available. The aim of the work described in this thesis was to analyze reactions of chlorambucil with 2’-deoxyribonucleosides and calf thymus DNA in aqueous buffered solution, at physiological pH, and to identify and characterize all adducts by using modern analyzing methods. Our research was also focused on the reactions of phenylacetic acid mustard with 2’-deoxynucleosides under similar conditions. A review of the literature consisting of general background of nucleic acids, alkylating agents and ultraviolet spectroscopy used to identify the purine and pyrimidine nucleosides, as well as the results from experimental work are presented and discussed in this doctoral thesis.

Als die alte Mutter

Soitinnus: lauluääni, piano.

Application of morphometry, static DNA ploidy analysis, and steroid receptor expression in diagnosis and prognosis of Libyan breast cancer

The aim of this study was to describe the demographic, clinicopathological, biological and morphometric features of Libyan breast cancer patients. The supporting value of nuclear morphometry and static image cytometry in the sensitivity for detecting breast cancer in conventional fine-needle aspiration biopsies were estimated. The findings were compared with findings in breast cancer in Finland and Nigeria. In addation, the value of ER and PR were evaluated. There were 131 histological samples, 41 cytological samples, and demographic and clinicopathological data from 234 Libyan patients. The Libyan breast cancer is dominantly premenopausal and in this feature it is similar to breast cancer in sub-Saharan Africans, but clearly different from breast cancer in Europeans, whose cancers are dominantly postmenopausal in character. At presention most Libyan patients have locally advanced disease, which is associated with poor survival rates. Nuclear morphometry and image DNA cytometry agree with earlier published data in the Finnish population and indicate that nuclear size and DNA analysis of nuclear content can be used to increase the cytological sensitivity and specificity in doubtful breast lesions, particularly when free cell sampling method is used. Combination of the morphometric data with earlier free cell data gave the following diagnostic guidelines: Range of overlap in free cell samples: 55 μm2 -71 μm2. Cut-off values for diagnostic purposes: Mean nuclear area (MNA) >54 μm2 for 100% detection of malignant cases (specificity 84 %), MNA < 72 μm2 for 100% detection of benign cases (sensitivity 91%). Histomorphometry showed a significant correlation between the MNA and most clinicopathological features, with the strongest association observed for histological grade (p <0.0001). MNA seems to be a prognosticator in Libyan breast cancer (Pearson’s test r = - 0.29, p = 0.019), but at lower level of significance than in the European material. A corresponding relationship was not found in shape-related morphometric features. ER and PR staining scores were in correlation with the clinical stage (p= 0.017, and 0.015, respectively), and also associated with lymph node negative patients (p=0.03, p=0.05, respectively). Receptor-positive (HR+) patients had a better survival. The fraction of HR+ cases among Libyan breast cancers is about the same as the fraction of positive cases in European breast cancer. The study suggests that also weak staining (corresponding to as few as 1% positive cells) has prognostic value. The prognostic significance may be associated with the practice to use antihormonal therapy in HR+ cases. The low survival and advanced presentation is associated with active cell proliferation, atypical nuclear morphology and aneuploid nuclear DNA content in Libyan breast cancer patients. The findings support the idea that breast cancer is not one type of disease, but should probably be classified into premenopausal and post menopausal types.

Libyan breast cancer: Health services and biology. Diagnosis delay and prognostic value of DNA pploidy, S-phase fraction, and Ki-67 and Bcl-2 immunohistochemistry

The aim of this study was to investigate the diagnosis delay and its impact on the stage of disease. The study also evaluated a nuclear DNA content, immunohistochemical expression of Ki-67 and bcl-2, and the correlation of these biological features with the clinicopathological features and patient outcome. 200 Libyan women, diagnosed during 2008–2009 were interviewed about the period from the first symptoms to the final histological diagnosis of breast cancer. Also retrospective preclinical and clinical data were collected from medical records on a form (questionnaire) in association with the interview. Tumor material of the patients was collected and nuclear DNA content analysed using DNA image cytometry. The expression of Ki-67 and bcl-2 were assessed using immunohistochemistry (IHC). The studies described in this thesis show that the median of diagnosis time for women with breast cancer was 7.5 months and 56% of patients were diagnosed within a period longer than 6 months. Inappropriate reassurance that the lump was benign was an important reason for prolongation of the diagnosis time. Diagnosis delay was also associated with initial breast symptom(s) that did not include a lump, old age, illiteracy, and history of benign fibrocystic disease. The patients who showed diagnosis delay had bigger tumour size (p<0.0001), positive lymph nodes (p<0.0001), and high incidence of late clinical stages (p<0.0001). Biologically, 82.7% of tumors were aneuploid and 17.3% were diploid. The median SPF of tumors was 11% while the median positivity of Ki-67 was 27.5%. High Ki-67 expression was found in 76% of patients, and high SPF values in 56% of patients. Positive bcl-2 expression was found in 62.4% of tumors. 72.2% of the bcl-2 positive samples were ER-positive. Patients who had tumor with DNA aneuploidy, high proliferative activity and negative bcl-2 expression were associated with a high grade of malignancy and short survival. The SPF value is useful cell proliferation marker in assessing prognosis, and the decision cut point of 11% for SPF in the Libyan material was clearly significant (p<0.0001). Bcl-2 is a powerful prognosticator and an independent predictor of breast cancer outcome in the Libyan material (p<0.0001). Libyan breast cancer was investigated in these studies from two different aspects: health services and biology. The results show that diagnosis delay is a very serious problem in Libya and is associated with complex interactions between many factors leading to advanced stages, and potentially to high mortality. Cytometric DNA variables, proliferative markers (Ki-67 and SPF), and oncoprotein bcl-2 negativity reflect the aggressive behavior of Libyan breast cancer and could be used with traditional factors to predict the outcome of individual patients, and to select appropriate therapy.

Der alte Soldat

Soitinnus: mieskuoro.

Heterologisen geenin DNA-sekvenssin optimointi sekä Fab-fragmentin DNA-sekvenssin vaikutus Escherichia colin kasvuun ja tuotto-ominaisuuksiin

Synonyymisillä kodoneilla tarkoitetaan kodoneja, jotka ovat erilaisia, mutta koodaavat samaa aminohappoa. Synonyymisten mutaatioiden, eli kodonin vaihtaminen samaa aminohappoa koodaavaan toiseen kodoniin, on pitkään ajateltu olevan yhdentekeviä, mutta synonyymit muutokset DNA-sekvenssissä voivat kuitenkin vaikuttaa esimerkiksi proteiinin laskostumiseen ja proteiinin toimintaan solussa. Eri organismit käyttävät synonyymisiä kodoneja eri frekvensseillä. Tätä ilmiötä kutsutaan kodonikäytön poikkeamaksi. Kodonikäytön poikkeamien on osoitettu olevan tärkein yksittäinen prokaryoottien geeniekspressioon vaikuttava tekijä, ja usein proteiineja on hankala tuottaa vieraassa isännässä, jos kodonikäytön poikkeamat ovat liian suuria. Erilaisia kodonioptimointistrategioita on kehitetty näiden ongelmien ratkaisemiseksi. Kodonikäyttöä optimoitaessa on otettava huomioon lisäksi erilaiset paikalliset muuttujat, jotka lisäävät optimoinnin monimutkaisuutta. Työn lähtökohtana oli synteettinen ihmisen vasta-aineen Fab-fragmentin geeni. Geeni oli optimoitu kahdella eri strategialla, jotka tuottivat eri DNA-sekvenssit, mutta saman aminohapposekvenssin. Toinen varianteista tuotti aktiivista Fab-fragmenttia, toinen ei. DNA-sekvenssin vaikutuksen tutkimiseksi, toimivan geenin osia korvattiin toimimattoman variantin vastaavalla osalla. Kaikkiaan seitsemän geenivariantin kykyä ilmentää Fab-fragmenttia sekä liukoisena proteiinina että filamenttifaagin pinnalla vertailtiin. Lisäksi tutkittiin varianttien vaikutusta isäntäsolun kasvukinetiikkaan. Muunneltuja variantteja verrattiin alkuperäiseen toimivaan varianttiin. Faagituotossa havaittiin Fab-fragmentin kevyen ketjun DNA-sekvenssin synonyymisten muutosten vaikuttavan faagien immunoreaktiivisuuteen. Erityisen olennainen oli kevyen ketjun vakioisen alueen muuttaminen, joka myös aiheutti 45 % laskun faagien kokonaismäärässä ja 54 % laskun faagien immunoreaktiivisuudessa. Liukoista proteiinia tuotettaessa kevyen ketjun vakioisen alueen muutos laski Fab-määrän tasolle, jota ei voitu mitata. Tämän lisäksi, avoimen lukukehyksen alun kodonien synonyymiset mutaatiot aiheuttivat 48-kertaisen laskun aktiivisen liukoisen proteiinin määrässä verrattuna alkuperäiseen toimivaan varianttiin.