Protein kinase CKII (i.e. casein kinase 11, CKII, NII) is expressed at a higher level in rapidly proliferating tissues and in solid human tumours (e. g. colorectal carcinomas) when compared to the corresponding nonneoplastic colorectal mucosa. This could be shown by (a) Western blotting of cellular extracts from solid tumours followed by immunostaining with an anti-CKII polyclonal antibody, (b) immunohistochemical staining of cells from tissue sections and (c) by activity measurements using the CKII-specific synthetic peptide (RRRDDDSDDD). The maximum observed activity in the colorectal carcinomas investigated was up to eightfold higher in the tumour specimens than in the non-neoplastic tissue (i.e. colorectal mucosa). The activity range was between 33 -350 U/ mg protein and in the case of colorectal mucosa 13 -106 U/mg protein. The amount of CKII determined in the individual tumours was in the range 0.4-1.6 nmol/g tissue.Casein kinase I1 (CKII) is found in the cytoplasm and in the nucleus. The latter form has also been called NII, although biochemical and biophysical data suggest that it is the same enzyme. The name is misleading inasmuch as the name casein kinase I1 was coined according to its test substrate which is casein.CKII is a ubiquitous protein kinase which is widely distributed in different eukaryotic cell types [I]. The level of enzyme activity has been shown to be elevated in transformed cells [2], in mitogen-stimulated lymphocytes [3], during mouse embryogenesis [4], during differentiation of 3T3-LI cells [5] and during progesterone-induced maturation of oocytes [6]. All these findings suggest a functional role for CKII in the regulation of cellular growth. In detail, CKII has been thought to play a role in the regulation of protein synthesis 141. By immunohistochemical studies, CKII was shown to be highly concentrated in the nucleolus [15, 161, i.e. in the same cellular compartment where some of its potential physiological substrates (RNA polymerase I, topoisomerases I and 11, and nucleolin) are located. These nucleolar proteins are involved in the first steps of cellular growth and CKII seems to be the key enzyme controlling the very first steps of proliferation at the level of rRNA synthesis. Here we demonstrate that CKII is elevated in human solid tumours when compared to nonneoplastic tissue of the same patient. The results are supported by immunohistochemical studies. In addition, we have shown that CKII is not exclusively elevated in tumour cells but also in normal tissue with high mitotic activity (e. g. colorectal mucosa).Thus the data presented are in good agreement with earlier studies, where elevated CKII activity was detected in tumour cell lines [2] and also in non-neoplastic tissue (e.g. during certain stages of embryogenesis) [4]. A comparison with the proliferation marker Ki67 [I71 also suggests that there may be a potential application for CKII as a marker protein for proliferation. MATERIALS AND METHODS Breast carcinomasA tumour and non-neoplastic tissue from a patient with an i...

Proteinase 3 (P3) is a serine proteinase present in the primary granules of neutrophils. We have investigated the expression of this protein in samples of bone marrow from healthy individuals and patients with different types of leukaemias by using immunocytochemical staining and flow cytometric quantitation. In normal bone marrow the enzyme was found in promyelocytes, myelocytes, metamyelocytes, band forms and polymorphonuclear neutrophils, correlating with the synthesis of neutrophil serine proteinases during myeloid maturation. No staining was found within the lymphoid, erythroid and megakaryocytic lineage. In the leukaemic samples, only those of acute myeloid and chronic myeloid leukaemia patients were labelled with the antiproteinase 3 antibody. Cases of acute lymphoblastic and chronic lymphocytic leukaemia, as well as other malignant lymphomas, were consistently negative, indicating that P3 may be used as a specific marker for the discrimination between myeloid and lymphoid leukaemias. In addition, immunoreactivity of myeloperoxidase (MPO) was investigated and the expression of P3 and MPO correlated with the French-American-British (FAB) classification. P3 was not detected in minimally differentiated M0 and M1 cases but was in predominantly labelled cells of M2 and M3 subtypes plus half of the M4 and one out of six M5 cases but not those of M6. These findings correspond to the differentiation stage in which P3 is expressed and stored in the primary granules. Therefore the enzyme may also be used as an adjunct to the classic morphological and cytochemical methods to elucidate further the stage at which the differentiation arrest of the leukaemic clone has occurred.

No abstract

No abstract

End-stage human heart failure is associated with changes in expression of steady-state messenger RNA (mRNA) levels. These changes correspond to alterations in protein levels and myocardial function and may have clinical implications regarding etiology, clinical state, or prognosis. However, analysis of mRNA levels in endomyocardial biopsies can be accomplished only by the quantitative polymerase chain reaction, which is difficult to standardize. The aim of the study was to evaluate whether the RNase protection assay is applicable to measure mRNAs of multiple genes simultaneously in small amounts of ventricular myocardium comparable to myocardial biopsies. Total RNA was prepared from left ventricular myocardium from terminally failing hearts with idiopathic (n=9) or ischemic cardiomyopathy (n=7) and from nonfailing control hearts (n=10). mRNA was measured by an optimized RNase protection assay for the beta1-adrenoceptor, the stimulatory G protein alpha-subunit (Gsalpha), phospholamban, the calcium ATPase of the sarcoplasmic reticulum (SERCA), beta-myosin heavy chain (beta-MHC), and the atrial natriuretic peptide (ANP). We extracted 10.7+/-2.1 microg total RNA from three myocardial biopsies taken in vitro. All of the six genes were measurable in duplicate in a total of 7 microg RNA. mRNAs of beta1-adrenoceptor, phospholamban, and SERCA were lower in failing than in nonfailing myocardium by 50%, 33%, and 42% respectively, whereas beta-MHC and Gsalpha mRNAs were unchanged. mRNA of ANP was expressed at high levels only in the failing myocardium, providing a highly specific and sensitive marker for discriminating nonfailing and failing hearts. A direct comparison with ANP and Gsalpha levels obtained by Northern blot analysis with 7.5 microg total RNA showed a good correlation between the two methods. The RNase protection assay is thus a suitable method for simultaneous measurements of multiple mRNA levels in human myocardial biopsies. Changes in mRNA levels closely reflected those identified by other methods using larger amounts of RNA. Increased myocardial ANP mRNA levels determined by the RNase protection assay may serve as a molecular marker of heart failure.