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Plasma from patients with iron overload resulting from idiopathic hemochromatosis contains nontransferrin-bound iron, measurable by the bleomycin, assay. During venesection therapy, the concentration of bleomycin iron declines in a way highly correlated with plasma ferritin concentrations. Even when patients had been venesected to give very low total plasma iron concentrations and high transferrin iron-binding capacity, bleomycin-detectable iron was still present at low concentrations. Bleomycin-detectable iron can stimulate damaging free radical reactions, and its persistence in plasma even after prolonged venesection might contribute to the tissue damage that results from iron overload.

Steroid binding assay using the dextran coated charcoal (DCC) method was applied to human tissues including tumors of the digestive organs, and the results were compared with those of enzymeimmunoassay (EIA) and immunocytochemical assay (ICA) with monoclonal antibody against human estrogen receptor of MCF-7 breast cancer cells. Using the DCC method, estrogen receptor activity was detected in 6 of 26 cases (23.1%) with gastric carcinoma, 3 of 16 hepatocellular carcinoma cases (18.8%), 1 of 3 gallbladder carcinoma cases (33.3%), and both of the 2 cases (100%) with normal liver tissue. However, using EIA, no ER activity was detected in any case. Moreover, ER positive cells were not found by immunohistochemical staining in the gastric carcinoma cases or in normal liver tissue, both of which showed ER activity by the DCC method. These results suggest that the estrogen receptor like material exists in cytosol of the human digestive tumors and normal liver tissue, but that the specificity of the antibodies against estrogen receptor molecules in these tumors may be different from that of the breast tumors.

Since freshly obtained acute lymphoblastic leukemia (ALL) cells rarely replicate spontaneously in vitro in a sustained way, development of a useful clonogenic assay for ALL blast progenitors is dependent on identifying the cellular growth requirements. Thus, marrows from 25 ALL cases were cultured in methylcellulose to determine the optimal conditions for cell growth. Blast colonies were confirmed as leukemic by morphology, cytochemistry, surface markers, and cytogenetics. Irradiated (7000 rads) normal peripheral blood feeder cells were an absolute requirement and produced number-dependent increases in ALL colonies; added growth factors enhanced the feeder cell effect. ALL cell-feeder cell contact was essential since their physical separation in a two-layer culture system drastically interfered with colony growth. Feeder cells from various donors, including new and relapsed cases of ALL, yielded colony numbers that differed widely when tested on the same marrow with and without added growth factor; thus, identification of a "good" feeder cell donor was key to an optimal assay. Neither recombinant interleukin-2 nor recombinant GM-CSF had ALL growth-promoting properties when tested alone or in combination but in the presence of feeder cells they moderately enhanced the feeder cell effect. The most effective growth factors were derived from cells exposed to phytohemagglutinin (PHA) for 72 h. In order of magnitude for colony growth-promoting activity, PHA-T cell conditioned medium (CM) was more stimulatory than PHA-blast cell CM followed by PHA-leukocyte CM; removal of PHA from CM by affinity chromotography did not alter the results. The most potent PHA-TCM was prepared from T-cells from a phlebotomized hemochromatosis patient; PHA-TCM from transfused thalassemia patients and normal donors were less active. Concanavalin-A blast cell CM had modest colony promoting properties whereas CM prepared with other B-cell mitogens and supernatants from ALL blasts in liquid culture had none. Our studies illustrate the complex and fastidious growth needs of ALL cells. The data have allowed us to refine a clonogenic blast progenitor assay that should facilitate study of proliferative properties of B and T lineage leukemias. The assay could be adapted further for detection of residual leukemia cells in marrow samples used for autologous transplantation, and in patients during complete hematological "remission."

In 129 cases, results from ER-ICA (Estrogen receptor immunocytochemical assay) applied to breast carcinoma biopsy smears and results from ER-EIA (Estrogen receptor enzyme immunoassay) applied to cytosols of breast carcinoma were compared to the results of ER(DCC), PR(DCC) and a cytomorphological grading. Using 500x magnification, the cases were graded visually in 7 groups, groups 1-4 without and groups 5-7 with nucleoli. Cases with high estrogen receptor values as determined by ER-EIA had the best chance of being detected also by the ER-ICA method. There is a relation between the visual nuclear grade from the Pappenheim stained specimens and ER-ICA and ER-EIA. The 48 cases without nucleoli had a mean ER-EIA-value of 100 fmol/mg (median = 59 fmol/mg). The 57 cases with nucleoli (groups 5-7) had a mean ER-EIA value of 37 fmol/mg (median = 14 fmol/mg). For 28 cases with prominent or multiple prominent nucleoli (groups 6-7) the mean ER-EIA value was only 15 fmol/mg (median = 5 fmol/mg). With increasing ER-EIA values we found decreasing average nuclear grades: 31 cases ER-EIA less than 10 fmol/mg, mean nuclear grade = 5.4; 34 cases ER-EIA greater than or equal to 10 less than 50 fmol/mg, mean nuclear grade = 4.6; 20 cases ER-EIA greater than or equal to 50 less than 100 fmol/mg, mean nuclear grade = 4.2; 19 cases ER-EIA greater than or equal to 100 fmol/mg, mean nuclear grade = 3.8. Our present data show clearly that there is a closer correlation of cytomorphology with ER-EIA than with ER(DCC) and PR(DCC). In the present study even ER-EIA alone correlated better with morphology than ER(DCC) and PR(DCC) taken together.

Chicken erythrocyte nuclei previously incubated separately with two novel mercury compounds (N-chloromercuribenzoyl)-biocytin and bis(p-(chloromercuribenzoyl))-[3H]lysine diamide) were digested with micrococcal nuclease and the digest products fractionated according to their solubility in 0.15 M NaCl and molecular size. The identity and quantitation of the chromatin fractions and proteins containing covalently bound mercury were determined by Western blotting, autoradiography, and scintillation counting. The most highly acetylated species of histone H3 in the 0.15 M NaCl-soluble polynucleosome fraction also contained the highest proportion of bound mercury. This fraction contains hyperacetylated core histones, is depleted in linker histones, and enriched in nonhistone proteins. Histone H3 in the 0.15 M NaCl-soluble mononucleosomes, which are unacetylated and lack linker histones, was 45% less labeled than histone H3 in the 0.15 M NaCl-soluble polynucleosome fraction. In the 0.15 M NaCl-insoluble polynucleosomes, which contain unacetylated histones and molar proportions of linker histones, histone H3 was 63% less labeled. Allowing for the differential abundance of these subfractions in the nucleus, the relative H3 reactivities are 50, 7, and 1 for 0.15 M NaCl-soluble polynucleosomes, mononucleosomes, and 0.15 M NaCl-insoluble polynucleosomes, respectively. Thus a gradation of reactivities exists which correlates with increasing hyperacetylation and linker histone depletion. High mobility group proteins 1 and 2, found in subnucleosome particles in the 0.15 M NaCl-soluble fraction, are extensively mercury-labeled. Distribution of histone acetyltransferase activity among salt- and size-resolved micrococcal nuclease produced fractions was almost 5-fold greater in the 0.15 M NaCl-soluble supernatant than in the 0.15 M NaCl-insoluble pellet. Furthermore, the acetyltransferase activity, which is tightly bound to undigested chromatin, is rapidly released by both micrococcal nuclease and DNase I. For short digestion times the enzyme is associated with the salt-soluble polynucleosomes, but at longer times of digestion the enzyme appears to be free from intact nucleosomes. The enzyme may be localized in the globin domain in erythrocytes and maintains that region in a hyperacetylated state which results in an altered linker histone binding reflected in a change in the reactivity of the usually inaccessible H3 cysteine 110.