Effect of cyclic AMP on chromatin-bound protein kinases in WI-38 fibroblasts stimulated to proliferate.

When resting confluent monolayers of WI-38 fibroblasts are stimulated to proliferate by serum, DNA synthesis begins to increase between 15-18 h after stimulation. Chromatin-bound protein kinase activity increases in stimulated cells within 1 h after the nutritional change, concomitant with an increase in the template activity of nuclear chromatin. Addition of dibutyryl 3' : 5'-cyclic adenosine monophosphate (dibutyryl cyclic) AMP to the stimulating medium inhibits the entrance of cells into S phase, but only if dibutyryl cyclic AMP (5-10(-4) M) is added before the onset of DNA synthesis. The increases in chromatin template activity and in the chromatin-bound kinase activity are not inhibited by dibutyryl cyclic AMP in the early hours after stimulation, but are completely inhibited after the 5th hour from the nutritional change. This seems to indicate that in stimulated WI-38 cells, dibutyryl cyclic AMP exerts its inhibitory action somewhere between 5 and 12 h after stimulation. A number of protein kinase activities were extracted from chromatin with 0.3 M NaCl and partially resolved on a phosphocellulose column. Two distinct peaks of protein kinase activity appeared to be markedly increased in WI-38 cells 6 h after serum stimulation. Both peaks of increased activity were inhibited by dibutyryl cyclic AMP in vivo. Adenosine, sodium butyrate and adenosine 5'-monophosphate (AMP) do not inhibit the increase in DNA synthesis nor the increase in protein kinase activity. The results suggest that stimulation of cell proliferation in confluent monolayers of WI-38 cells causes an increase (or the new appearance) of certain chromatin-bound protein kinases, and that this increase is inhibited by cyclic AMP in vivo.

A micromethod to culture granulocyte colonies from human bone marrow in agar-containing glass capillaries.

A micromethod was developed to grow granulocytic colonies from human bone marrow in agar-containing glass capillary tubes. Treatment of the bone marrow samples and the culture conditions (type and quantity of serum, CSF, cell seeding density) were optimized. Up to 60 colonies were obtained from 3.5 x 10(4) nucleated cells seeded into 50 microliter of total incubation medium/capillary with horse serum (13%) and leukocyte and partially purified bovine lung conditioned medium as CSF (17 and 3%, respectively). The micromethod requires less culture materials (about 1/20), cells, CSF and less time for colony counting, but higher cell densities for seeding, resulting in an increased sensitivity for drug or factor testing. Colony morphology can be easily examined. The micromethod offers further advantages, e.g. quantitation by light scattering densitometry, and hence seems suitable for clinical investigations.

Increased RNA synthesis in nuclear monolayers of WI-38 cells stimulated to proliferate.

Nuclear monolayers of WI-38 cells prepared by the method of Tsai and Green were used to determine RNA synthesis in isolated nuclei in situ. In nuclear monolayers, incorporation of [(3)H]UTP into RNA is dependent on the presence of the other three nucleotide triphosphate and is abolished by actinomycin D. The extent of RNA synthesis under these conditions was measured in density-inhibited WI-38 human diploid fibroblasts at various intervals after cell proliferation was stimulated by a change of medium.RNA synthesis increases 15 min after the nutritional change and reaches a peak at 18 hr, which is also the peak of DNA synthesis. Thereafter RNA synthesis declines. Essentially similar results are obtained whether the endogenous RNA polymerase or a bacterial polymerase is used. Replacement of the stimulating medium by conditioned medium stops the increase in RNA synthesis that occurs in cultures subject to continuous stimulation. Finally, RNA synthesis in nuclear monolayers, using the endogenous RNA polymerase, occurs by chain elongation only, while re-initiation occurs with the bacterial RNA polymerase.

[Chemotherapy of metastasizing colonic carcinomas with nitrosourea compounts. Retrospective analysis]. / Chemotherapie des metastasierten Kolonkarzinoms mit Nitroseharnstoffen.

35 patients with advanced, and previously untreated, colorectal cancer underwent therapy with a combination of 5-fluorouracil, vincristine, DTIC, or one of the nitrosoureas, BCNU or methyl-CCNU, respectively. No complete remission was achieved. 33% of all cases resulted in partial remission and lasted for approximately 3--4 months. Complications were perilous such as neutro- and thrombocytopenia. The overal quality of life was poor. Though several parameters influence these results, we think that the above-listed nitrosoureas do not add substance to the chemotherapy of colorectal cancer.

Ferritin in bone marrow and serum in iron deficiency and iron overload.

Nonheme iron and ferritin in the bone marrow and serum ferritin was investigated in patients with iron deficiency anaemia or iron overload. As controls served patients without any disturbance of the iron metabolism. There is a precise correlation between the nonheme iron and ferritin in the bone marrow of patients with and without disturbance of iron metabolism. A correlation was also found between the ferritin in the bone marrow and the serum. Nonheme iron and ferritin in the bone marrow and serum ferritin was decreased in patients with iron deficiency anaemia. Conversely, the same parameters were increased in patients with iron overload.

Isolation of transcriptionally active chromatin from mammalian nucleoli.

Nucleoli isolated from HeLa cells are functionally active but contain large amounts of RNA and proteins (RNA/DNA ratio 1:1; protein/DNA ratio 7:1). We have isolated from the nucleolus a DNA-protein complex that has the characteristics of nucleolar chromatin (RNA/DNA ratio less than 0.05:1; protein/DNA ratio 1.7:1). This nucleolar chromatin has most of the transcriptional activity of the intact nucleolus and, as assayed by circular dichroism and dye binding, has largely preserved its structure. The isolation of a transcriptionally active, fragment of chromatin, which constitutes only a small part of the total genome and codes for only one recognizable product, offers several advantages for the study of chromatin structure and function.