Activity of RNA polymerase I transcription factor UBF blocked by Rb gene product.

The protein encoded by the retinoblastoma susceptibility gene (Rb) functions as a tumour suppressor and negative growth regulator. As actively growing cells require the ongoing synthesis of ribosomal RNA, we considered that Rb might interact with the ribosomal DNA transcription apparatus. Here we report that (1) there is an accumulation of Rb protein in the nucleoli of differentiated U937 cells which correlates with inhibition of rDNA transcription; (2) addition of Rb to an in vitro transcription system inhibits transcription by RNA polymerase I; (3) this inhibition requires a functional Rb pocket; and (4) Rb specifically inhibits the activity of the RNA polymerase I transcription factor UBF (upstream binding factor) in vitro. This last observation was confirmed by affinity chromatography and immunoprecipitation, which demonstrated an interaction between Rb and UBF. These results indicate that there is an additional mechanism by which Rb suppresses cell growth, namely that Rb directly represses transcription of the rRNA genes.

Translocation of retinoblastoma protein associated with tumor cell growth inhibition.

Immunocytochemistry revealed an association between growth inhibition and a translocation of retinoblastoma protein (pRB) to the nucleoli of U-2 osteosarcoma cells inhibited in their growth by the negative growth factor NCPI (natural cell proliferation inhibitor). Similar phenomenon was demonstrated by Western blot analysis and immunocytochemistry in U937 leukemic cells inhibited in their growth and induced to differentiate by 12-O-tetradecanoylphorbol acetate (TPA). Total nuclear extract of control U937 cells gave an intense 110 KD band, while total nuclear extract of TPA treated cells produced an intense 60 KD band and a weak 110 KD band. No bands were observed for the purified nucleoli of control cells, but the purified nucleoli of TPA treated cells produced a 60 KD band. These results suggest that in the process of cell growth inhibition and differentiation, specific proteolysis of pRB and its translocation to the nucleolus may occur.

Increase in protein kinase C activity is associated with human fibroblast growth inhibition.

Protein kinase C (PKC) activity and DNA synthesis were measured in human fetal bone marrow fibroblasts following treatment with tumor necrosis factor alpha (TNF alpha) (500 U/ml) or conditioned media containing natural cell proliferation inhibitor (CM-NCPI). Treatment with TNF alpha led to growth stimulation (120 +/- 7% of control in 24 h, 141 +/- 6% in 72 h). At the same time particulate PKC activity diminished, reaching 55 +/- 8% of control in 24 h and remaining at this level at 72 h. CM-NCPI treatment of the cells resulted in a decrease in DNA synthesis (by 39 +/- 6% in 2 h, by 58 +/- 5% in 24 h, and by 78 +/- 8% in 72 h). This was accompanied by a significant rise in particulate PKC activity which increased over 3-fold in 2 h, over 5-fold in 24 h, and up to 11-fold in 72 h. This 11-fold elevation was maintained after 2 week exposure of the fibroblasts to CM-NCPI. The PKC inhibitor neomycin abolished CM-NCPI induced growth inhibition, whereas PKC activator 12-O-tetradecanoylphorbol 13-acetate intensified it. These results suggest that CM-NCPI acts as PKC activator and that negative growth regulation by extracellular agents may involve stimulation of PKC activity.

Fibroblast growth inhibitor.

A new cytokine has been recognized in the conditioned media (CM) of freshly isolated acute myelocytic leukemia cells, cultured with 12-0-tetradecanayl phorbol acetate (TPA) 10(-8)M. The fraction with 70,000 MW was separated from CM by ammonium sulfate precipitation, ion-exchange cation and anion chromatography, and Sephadex G-200 gel filtration. It was a fibroblast growth inhibitor (FGI). This substance stopped fetal and skin (MALME 3 line) fibroblast propagation. The cytostatic effect was reversible on removal of FGI. At the same time, FGI did not inhibit macrophage proliferation. The fraction stimulated formation of monocytic and granulocytic colonies altered the phenotype of human U-2 osteosarcoma cells grown from epithelial-like to fibroblast-like cells, and stimulated differentiation of leukemic cells along the macrophage path. Some cells of promyelocytic leukemia line HL-60, grown in the presence of FGI, were stimulated to differentiate and some underwent lysis. The response to FGI of cells from different patients varied.

Electrophoretic characteristics of macro creatine kinase type 2 in serum.

We have studied the electrophoretic characteristics of a creatine kinase (CK, EC 2.7.3.2) isoenzyme, macro creatine kinase type 2 (MCK-2), in the serum of 42 patients with carcinoma of the colon. The patients could be divided into three subgroups by the intensity of the MCK-2 band (nine high, nine medium, and 24 low intensity), but we detected no correlation between the isoenzyme activity and the stage of the cancer. Inhibition of the activity of the CK-M subunit by antibody to CK-M allowed more sensitive detection of MCK-2 in serum and revealed that MCK-2 may be obscured by CK-MM. Moreover, there may be major discrepancies between the amount of MCK-2 seen on electrophoresis and the apparent total CK.

Macro creatine kinase type 2 in human colonic tissues.

Macro creatine kinase type 2 (MCK-2) is a promising new tumour marker for carcinoma of the colon. We have purified it from tumour tissue, from serum and, for the first time, from normal colonic epithelium and identified it in a tissue culture line of colon cancer cells. The molecular weight of each preparation, regardless of the source, was similar. Its consistent presence in tumours and in normal colon, contrasted to its variable appearance in serum, implies that there are other factors affecting its release into, or removal from, the circulation.

Macrocreatine kinase type 2 in the serum of patients with tumors and with nonmalignant colonic diseases.

The macrocreatine kinase type 2 isoenzyme (MCK-2) was investigated as a marker for colonic cancer. It was sought in 252 serum samples from 231 patients: 69 with active colonic cancer, 49 in whom colonic cancer had been successfully resected, 58 with nonmalignant diseases of the colon, and 76 patients immediately following colonic surgery. MCK-2 was detected in the serum of 39 of the patients with cancer (57%) and in one patient with diverticulitis. MCK-2 and carcinoembryonic antigen (CEA) were both measured in 47 colonic cancer patients. Both markers were detected in 19 cases, MCK-2 alone in eight and CEA alone in eight. We conclude that MCK-2 is a promising tumor marker for carcinoma of the colon and that its value might be complementary to that of CEA.

Variations in carcinoembryonic antigen localization in tumors of the colon.

The localization of carcinoembryonic antigen (CEA) in 78 tumors of the human colon was studied with the use of antibody to CEA in an indirect Coons test. The neoplasms differed morphologically and could be divided into two types: a) tissues in which CEA was localized in cells, and b) tissues in which malignant cells secreted CEA in greater or lesser quantity. In the first type of tissue, five forms of cells were observed: goblet-like, small vacuolar, limbic, those with intracellular accumulation of antigen, and diffuse. Tumor tissue secreting CEA could also be divided into five forms: holocrine-like, aprocrine-like, intraglandular, apical-basal, and lacunar-infiltrative. The change in CEA localization in pathologically altered cells were designated 'antigenic translocation."