Katja Porvari

Differentially expressed genes in two LNCaP prostate cancer cell lines reflecting changes during prostate cancer progression.

Prostate cancer tends to become transformed to androgen-independent disease over time when treated by androgen-deprivation therapy. We used two variants of the human prostate cancer cell line LNCaP to study gene expression differences during prostate cancer progression to androgen-independent disease. Production of prostate-specific antigen was regarded as a marker of androgen-dependence and loss of prostate-specific antigen was regarded as a marker of androgen-independence. mRNA from both cell lines was used for cDNA microarray screening. Differential expression of several genes was confirmed by Northern blotting. Monoamine oxidase A, an Expressed Sequence Tag (EST) similar to rat P044, and EST AA412049 were highly overexpressed in androgen-dependent LNCaP cells. Tissue-type plasminogen activator, interferon-inducible protein p78 (MxB), an EST similar to galectin-1, follistatin, fatty acid-binding protein 5, EST AA609749, annexin I, the interferon-inducible gene 1–8U, and phospholipase D1 were highly overexpressed in androgen-independent LNCaP cells. All studied genes had low or no expression in PC-3 cells. The EST similar to rat P044, the EST similar to galectin-1, follistatin, annexin I, and the interferon-inducible gene 1–8U were also expressed in benign prostatic hyperplasia tissue. The Y-linked ribosomal protein S4, Mat-8, and EST AA307912 were highly expressed in benign prostatic hyperplasia tissue. Additionally, both confirmation of differential expression in Northern blots and in situ hybridization were carried out for monoamine oxidase A, the EST similar to rat P044, the EST similar to galectin-1, fatty acid-binding protein 5, and the interferon-inducible gene 1–8U. We identified several potential prostate cancer markers, indicating that the method used is a useful tool for the screening of cancer markers, but other methods, such as in situ hybridization, are needed to further investigate the observations.

SEVERAL GENES ENCODING RIBOSOMAL PROTEINS ARE OVER-EXPRESSED IN PROSTATE-CANCER CELL LINES: CONFIRMATION OF L7a AND L37 OVER-EXPRESSION IN PROSTATE-CANCER TISSUE SAMPLES

A cDNA library specific for mRNA over‐expressed in prostate cancer was generated by subtractive hybridization of transcripts originating from prostatic hyperplasia and cancer tissues. cDNA encoding ribosomal proteins L4, L5, L7a, L23a, L30, L37, S14 and S18 was found to be present among 100 analyzed clones. Levels of ribosomal mRNA were significantly higher at least in one of the prostate‐cancer cell lines, LNCaP, DU‐145 and PC‐3, than in hyperplastic tissue, as determined by slot‐blot hybridization. Furthermore, L23a‐ and S14‐transcript levels were significantly elevated in PC‐3 cells as compared with those in the normal prostate epithelial cell line PrEC. Generally, dramatic changes in the mRNA content of the ribosomal proteins were not detected, the most evident over‐expression being that of L37 mRNA, which was 3.4 times more abundant in LNCaP cells than in hyperplastic prostate tissue. The over‐expression of L7a and L37 mRNA was confirmed in prostate‐cancer tissue samples by in situ hybridization. Elevated cancer‐related expression of L4 and L30 has not been reported, but levels of the other ribosomal proteins are known to be increased in several types of cancers. These results therefore suggest that prostate cancer is comparable with other types of cancers, in that a larger pool of some ribosomal proteins is gained during the transformation process, by an unknown mechanism. Int. J. Cancer 78:27–32, 1998.© 1998 Wiley‐Liss, Inc.

The TMPRSS2 gene encoding transmembrane serine protease is overexpressed in a majority of prostate cancer patients: Detection of mutated TMPRSS2 form in a case of aggressive disease

The serine protease TMPRSS2 gene expression was studied by in situ hybridization using benign prostatic hyperplasia and prostate cancer tissue samples from 32 patients. Expression of TMPRSS2 gene was higher in cancer cells than that in benign cells in 84% of the specimens containing both benign and malignant tissues. The TMPRSS2 mRNA level was significantly increased in poorly differentiated (p = 0.014, n = 7) and untreated (p = 0.022, n = 13) primary prostate adenocarcinomas compared to benign tissues. In addition, androgen‐deprivation therapy significantly decreased the expression of TMPRSS2 in benign prostate tissue (p = 0.07), which is in accordance with the androgen‐inducible expression of the gene. The gene copy number of TMPRSS2, analyzed by competitively differential PCR, was duplicated in the malignant cells of about 38% of the prostate cancer patients analyzed. Thus, the increase in the gene copy number is probably not the primary reason for the detected overexpression of the TMPRSS2 gene. Mutations in the TMPRSS2 gene were screened using DNA isolated from paraffin‐embedded prostate cancer tissues from 9 patients with aggressive prostate cancer and from 9 patients with nonaggressive disease. Thirteen exons covering the coding region were checked using enzymatic mutation detection and direct sequencing. One patient with aggressive prostate cancer carried a deletion and a stop codon in exon 11, leading to inactivation of the serine protease domain in TMPRSS2.

O-Glycosylation Regulates LNCaP Prostate Cancer Cell Susceptibility to Apoptosis Induced by Galectin-1

Resistance to apoptosis is a critical feature of neoplastic cells. Galectin-1 is an endogenous carbohydrate-binding protein that induces death of leukemia and lymphoma cells, breast cancer cells, and the LNCaP prostate cancer cell line, but not other prostate cancer cell lines. To understand the mechanism of galectin-1 sensitivity of LNCaP cells compared with other prostate cancer cells, we characterized glycan ligands that are important for conferring galectin-1 sensitivity in these cells, and analyzed expression of glycosyltransferase genes in galectin-1-sensitive, prostate-specific antigen-positive (PSA(+)) LNCaP cells compared with a galectin-1-resistant PSA(-) LNCaP subclone. We identified one glycosyltransferase, core 2 N-acetylglucosaminyltransferase, which is down-regulated in galectin-1-resistant PSA(-) LNCaP cells compared with galectin-1-sensitive PSA(+) LNCaP cells. Intriguingly, this is the same glycosyltransferase required for galectin-1 susceptibility of T lymphoma cells, indicating that similar O-glycan ligands on different polypeptide backbones may be common death trigger receptors recognized by galectin-1 on different types of cancer cells. Blocking O-glycan elongation by expressing alpha2,3-sialyltransferase 1 rendered LNCaP cells resistant to galectin-1, showing that specific O-glycans are critical for galectin-1 susceptibility. Loss of galectin-1 susceptibility and synthesis of endogenous galectin-1 has been proposed to promote tumor evasion of immune attack; we found that galectin-1-expressing prostate cancer cells killed bound T cells, whereas LNCaP cells that do not express galectin-1 did not kill T cells. Resistance to galectin-1-induced apoptosis may directly contribute to the survival of prostate cancer cells as well as promote immune evasion by the tumor.

Expression of transmembrane serine protease TMPRSS2 in mouse and human tissues.

The purpose of this study was to clarify the expression of TMPRSS2 in mice during development and to compare the tissue distribution of the transcripts in adult mouse and human tissues. Mouse TMPRSS2 cDNA was cloned; the predicted amino acid sequence contains 490 residues sharing 81.4% similarity with human TMPRSS2. According to northern blots, mouse TMPRSS2 is expressed mainly in the prostate and kidney, while human TMPRSS2 is expressed in the prostate, colon, stomach, and salivary gland. In situ hybridization analyses of mouse embryos and adult tissues revealed that TMPRSS2 was expressed in the epithelia of the gastrointestinal, urogenital, and respiratory tracts. Expression was very selective and constant after the gene was turned on during development. Expression of TMPRSS2 was localized in the luminal epithelial cells of the mouse and human prostate. The information presented here will be useful in further studies regarding the function and physiological significance of TMPRSS2. Copyright

17β-Hydroxysteroid dehydrogenases and cancers☆

Abstract 17β-Hydroxysteroid dehydrogenases (17HSDs) catalyze the interconversions between active 17β-hydroxysteroids and less-active 17-ketosteroids thereby affecting the availability of biologically active estrogens and androgens in a variety of tissues. The enzymes have different enzymatic properties and characteristic cell-specific expression patterns, suggesting differential physiological functions for the enzymes. Epidemiological and endocrine evidence indicate that estrogens play a key role in the etiology of breast cancer while androgens are involved in mechanisms controlling the growth of prostatic cells, both normal and malignant. Recently, we have developed, using LNCaP prostate cancer cell lines, a cell model to study the progression of prostate cancer. In the model LNCaP cells are transformed in culture condition to more aggressive cells, able to grow in suspension cultures. Our results suggest that substantial changes in androgen and estrogen metabolism occur in the cells during the process. These changes lead to increased production of active estrogens during transformation of the cells. Data from studies of breast cell lines and tissues suggest that the oxidative 17HSD type 2 may predominate in human non-malignant breast epithelial cells, while the reductive 17HSD type 1 activity prevails in malignant cells. Deprivation of an estrogen response by using specific 17HSD type 1 inhibitors is a tempting approach to treat estrogen-dependent breast cancer. Our recent studies demonstrate that in addition to sex hormone target tissues, estrogens may be important in the development of cancer in some other tissues previously not considered as estrogen target tissues such as colon. Our data show that the abundant expression of 17HSD type 2 present in normal colonic mucosa is significantly decreased during colon cancer development.

Increased adrenaline to noradrenaline ratio is a superior indicator of antemortem hypothermia compared with separate catecholamine concentrations.

Abstract:  The significance of urinary catecholamines and small gastric mucosal bleedings, Wischnewsky’s spots, in postmortem diagnosis of hypothermia deaths was evaluated. Autopsy cases (n = 358) were divided into hypothermia, suspected hypothermia, and control groups. The catecholamine levels did not correlate with the length of the postmortem period. The adrenaline to noradrenaline ratio was most effective in detecting hypothermia (68.9% sensitivity, 78.1% specificity). The median adrenaline concentrations were significantly higher in hypothermia than in control groups. The control group containing mostly sudden cardiac deaths with no cold exposure had a noradrenaline level comparable to the hypothermia groups. The sensitivity and specificity of determining Wischnewsky’s spots in hypothermia deaths were 63.9% and 88.3%, respectively. The adrenaline to noradrenaline ratio is more suitable in proving antemortem cold stress than either of these independently, and its diagnostic value is comparable to that of Wischnewsky’s spots.

Extreme obesity and associated cardiovascular disease verified at autopsy: time trends over 3 decades.

Abstract Extreme obesity is a strong predictor of premature death, but the prevalence of cardiovascular disease in morbidly obese populations is largely unknown. The aim of the present study was to find out whether there has been an increase in extreme obesity with body mass index 40.0 kg/m2 or greater in medicolegal autopsy material in a known geographical area in Finland during a period of 3 decades and to examine the prevalence and time trends of associated cardiovascular disease in this obesity category. Autopsy reports of 235 cases examined in 1975 to 2006 were analyzed. The number of extremely obese individuals increased from 0.6% of the yearly amount of autopsies in the 1970s and 1980s to 2.8% and 2.5% in 2005 and 2006, respectively. The most frequent cause of death was cardiomyopathy or cardiomegaly (28.9%), followed by coronary heart disease (24.3%). Either coronary arteries were lesion-free, or only fatty streaks had been observed in 46.8% of the women and in 43.1% of the men. No significant changes in the average body mass index or severity of coronary atherosclerosis were observed. Younger individuals younger than 40 years began to appear more often after 1995. An increased trend of extreme obesity in a region where autopsy frequency is high may refer to a general increase of this obesity category. A large number of extremely obese people are resistant to coronary atherosclerosis, but cardiac hypertrophy may be accompanied by several mechanisms leading to sudden death even among the youngest extremely obese individuals.

Theoretical investigations of prostatic acid phosphatase

The phosphotyrosyl protein phosphatase activity of prostatic acid phosphatase (PAP) has been well established. It has also been suggested that PAP partly regulates the activity of growth factor receptors by dephosphorylating the autophosphorylysable tyrosines in them. We studied the binding of the peptides from epidermal growth factor receptor (EGFR) and its homolog (ErbB‐2), corresponding to their autophosphorylation sites, to PAP using theoretical modeling and molecular dynamics (MD) simulation methods. Nine different peptides, each with a phosphotyrosine residue, were docked on human PAP. The binding energies of these peptide–PAP complexes were calculated theoretically and compared to experimentally obtained affinities. The peptide AceDNLpYYWDNH2 from ErbB‐2(1197–1203) showed the most favorable free energy of binding when estimated theoretically. The results demonstrate that the presence of another tyrosine residue proximate to C‐terminal of autophosphorylysable Tyr enhances the binding affinity considerably. The presence of a bulky group instead prevents the binding, as is observed in case of peptide AceNLYpYWDQNH2 which failed to bind, both in theoretical calculations and experiments. Thus we demonstarted that PAP could potentially bind to EGFR and Erbb‐2 and dephosphorylate them. Thus it could be involved in the regulation of the function of such receptors. In addition, complexes of a peptide from AngiotensinII and phosphotyrosine(pY) with human PAP were also modeled. The effects of different protonation states of the titratable active site residues on ligand (pY) binding have also been investigated. For a favorable binding His12 and Asp258 should be neutral, His257 should be positively charged and the phosphate group of the ligand should be in PO  43− state. Furthermore, the analysis of protein motion as observed during simulations suggests the loop–loop contact in the PAP dimer to be of importance in cooperativity. Proteins 2005.

Adventitial macrophage and lymphocyte accumulation accompanying early stages of human coronary atherogenesis

BACKGROUND Atherosclerosis is considered a chronic inflammatory disease of the entire arterial wall, including the adventitia. Advanced coronary lesions with lipid cores are associated with adventitial inflammation, but the early inflammatory process in human coronary adventitia is largely unknown. We hypothesized that adventitial inflammatory cell infiltration accompanies the early stages of atherogenesis in human coronary arteries, and it is synchronous with the inflammatory process in the intima. METHODS Coronary artery samples were obtained from 111 forensic autopsy cases aged from 7 to 25 years. Adventitial and intimal macrophages, T lymphocytes and B lymphocytes, and intimal microvessels were detected by immunohistochemical methods and quantified by computerized image analysis. Body height, weight, waist circumference, and the size of mesenteric and omental fat depots were measured. RESULTS Adventitial densities of macrophages and T lymphocytes were significantly higher in arteries showing intimal xanthomas than in cases with only scattered intimal macrophages. The xanthoma group also had significantly higher body mass index and larger visceral fat depots. Highest densities of all adventitial cell types were seen in intermediate lesions and fibroatheromas. There were significant positive correlations between intimal and adventitial densities of T cells and B cells in the groups with or without intimal xanthomas, but the positive correlation between intimal and adventitial macrophages was significant only in the group without xanthomas. CONCLUSIONS Adventitial immune-inflammatory cell accumulation accompanies the early stages of coronary atherogenesis in young individuals, and lymphocyte accumulation seems to be synchronous in the intima and adventitia. Macrophage accumulation is also synchronous before xanthomas are seen.