Summary.We studied X-chromosome inactivation patterns in blood cells from normal females in three age groups: neonates (umbilical cord blood), 25-32 years old (young women group) and >75 years old (elderly women). Using PCR, the differential allele methylation status was evaluated on active and inactive X chromosomes at the human androgen receptor (HUMARA) and phosphoglycerate kinase (PGK) loci. A cleavage ratio (CR) у 3·0 was adopted as a cut-off to discriminate between balanced and unbalanced X-chromosome inactivation. In adult women this analysis was also performed on hair bulbs. The frequency of skewed X-inactivation in polymorphonuclear (PMN) cells increased with age: CR у 3·0 was found in 3/36 cord blood samples, 5/30 young women and 14/31 elderly women.Mathematical analysis of patterns found in neonates indicated that X-chromosome inactivation probably occurs when the total number of haemopoietic stem cell precursors is 14-16. The inactivation patterns found in T lymphocytes were significantly related to those observed in PMNs in both young (P < 0·001) and elderly women (P < 0·01). However, the use of T lymphocytes as a control tissue for distinguishing between skewed inactivation and clonal proliferation proved to be reliable in young females, but not in elderly women, where overestimation of the frequency of clonal myelopoiesis may appear.

The plant toxin saporin is a ribosome-inactivating protein which inhibits protein synthesis and growth of both normal and tumour cells. Its cytotoxic activity can be increased by coupling with antibodies recognizing cell surface antigens. In this work we performed experiments to test the hypothesis that saporin induces cell death via apoptosis. Exposure to saporin induced apoptosis in different cellular models, such as human peripheral blood B lymphocytes and neutrophils, in the Daudi B-cell line, and in the haemopoietic cell lines HL-60 and TF-1. This was indicated by: (a) the appearance of typical morphological features such as chromatin condensation, nuclear fragmentation and blebbing of plasma membranes: (b) DNA degradation into oligonucleosomal fragments: (c) the appearance of apoptotic cells on DNA flow cytometry as a cell population with reduced DNA content (A0 region). The fraction of cells showing features of apoptosis ranged from 19 +/- 5% for TF-1 cells to 35 +/- 8% for neutrophils. In experiments with normal peripheral blood B lymphocytes or with Daudi cells, we compared the activity of native saporin with that of an immunotoxin hybrid molecule obtained by binding the toxin to two bispecific antibodies recognizing both saporin and the B lymphocyte-specific antigen CD22 (Sap/BsAb complexes). Saporin bound to the antibodies was 2-3 logs more effective than native saporin in inducing apoptosis, with maximal inhibitions being observed at concentrations of 10(-6) M for native saporin and 10(-9)-10(-8) M for the hybrid molecules. These findings indicate that treatment with saporin results in apoptosis of target cells and suggest that this may be relevant to the therapeutic use of saporin-containing immunotoxins. In fact, if used in vivo as an immunotoxin, its cytotoxic activity could be devoid of more extensive and non-specific tissue damaging effects as would be the case if saporin induced necrosis of target cells.

A number of experimental observations suggest that the proto-oncogene c-ab1 participates in the regulation of hematopoietic cell growth. We used an antisense strategy t o study the relationship between c-ab1 expression and hematopoietic cell proliferation and differentiation. Purified normal human bone marrow-derived CD34+ cells were obtained by immunomagnetic selection and incubated with 18-base-unmodified antisense oligodeoxynucleotides complementary t o t h e first six codons of the two alternative first exons of c-&l, la and lb. At the end of incubation, an aliquot of cells was assayed for clonogenic growth and the remainder was used for flow cytometric analyses. Cell kinetics were evaluated by means of both single parameter DNA and bivariate DNA/bromodeoxyuridine (BrdU) flow cytometry. Apoptosis was routinely studied by DNA flow cytometric analysis and, in some cases, also through DNA agarose gel electrophoresis for detection of oligonucleosomal DNA fragments. Expression of differentiation markers was studied by flow cytometry. Exposure t o antisense oligonucleotides specifically inhibited the accumulation of c-ab1 mRNA in CD34' cells. Preincubation with the c-ab1 antisense oligomers reduced the proportion of cells in S-phase from 19%