We evaluated different culture conditions to obtain a lineage-selected proliferation of clonogenic megakaryocytic progenitors (MP). In low-density (LD) or CD34+ cell cultures, the best results were obtained in serum-free medium in the presence of megakaryocyte growth and development factor, stem cell factor, interleukin-3 (IL-3), IL-6, IL-11, FLT-ligand, and macrophage inflammatory protein-1α. In paired studies, expansion of LD cells was less effective than expansion of CD34+ cells, and pre-enrichment of CD34+ cells using negative depletion of lineage-positive cells produced significantly larger quantities of MP than pre-enrichment using positive selection. MP proliferation peaked on day 7 in culture, and an 8- ± 5-fold expansion of CD34+/CD61+ cells, a 17- ± 5-fold expansion of colony-forming units-megakaryocytes, and a 58- ± 14-fold expansion of the total number of CD61+ cells was obtained. In a feasibility clinical study, 10 cancer patients (8 with breast cancer and 2 with non-Hodgkin's lymphoma) undergoing autologous peripheral blood progenitor cell (PBPC) transplant received MP generated ex vivo (range, 1 to 21 × 105/kg CD61+ cells) together with unmanipulated PBPC. Eight patients received a single allogeneic platelet transfusion, whereas platelet transfusion support was not needed in 2 of the 4 patients receiving the highest doses of cultured MP. This result compares favorably with a retrospective control group of 14 patients, all requiring platelet transfusion support. Adverse reactions or bacterial contamination of cell cultures have not been observed. In conclusion, MP can be expanded ex vivo and safely administered to autologous transplant recipients. Further clinical trials will indicate the reinfusion schedule able to consistently abrogate the need for allogeneic platelet transfusion support in autologous transplantation.

DNA topoisomerases have been proposed as the proteins involved in the formation of the DNA-protein cross-links detected after ultraviolet light (UV) irradiation of cellular DNA. This possibility has been investigated by studying the effects of UV-induced DNA damage on human DNA topoisomerase I action. UV lesions impaired the enzyme's ability to relax negatively supercoiled DNA. Decreased relaxation activity correlated with the stimulation of cleavable complexes. Accumulation of cleavable complexes resulted from blockage of the rejoining step of the cleavage-religation reaction. Mapping of cleavage sites on the pAT153 genome indicated UV-induced cleavage at discrete positions corresponding to sites stimulated also by the topoisomerase I inhibitor camptothecin, except for one. Subsequent analysis at nucleotide level within the sequence encompassing the UV-specific cleavage site revealed the precise positions of sites stimulated by camptothecin with respect to those specific for UV irradiation. Interestingly, one of the UV-stimulated cleavage sites was formed within a sequence that did not contain dimerized pyrimidines, suggesting transmission of the distortion, caused by photodamage to DNA, into the neighboring sequences. These results support the proposal that DNA structural alterations induced by UV lesions can be sufficient stimulus to induce cross-linking of topoisomerase I to cellular DNA.Cyclobutane pyrimidine dimers (CPDs) 1 and (6 -4) photoproducts are the most prevalent lesions produced in DNA by ultraviolet (UV) light (1). However, pyrimidine dimers are not the only photochemical effect of UV light on cellular DNA. It has been shown that UV radiation induces also the formation of DNA-protein cross-links (2-4). Proteinase K treatment abolishes the cross-linking effect and reveals the presence of cryptic DNA strand breaks. Since this cross-linking is partially repaired, it has been suggested that this non-dimer DNA damage may play an important role in the biological effect of UV radiation (2, 4). DNA topoisomerases have been proposed as possible candidates for the protein(s) involved in UV-induced DNAprotein cross-linking (4). This proposal is consistent with the formation of transient single and double strand breaks during DNA topoisomerase reactions, with covalent attachment of the enzymes to one terminus of the DNA nick (reviewed in Ref. 5).DNA topoisomerases are ubiquitous enzymes involved in a number of crucial cellular processes, including replication, transcription, and recombination. A relationship between cellular responses to DNA damage and topoisomerases has been proposed (6 -9). The catalytic cycle of DNA topoisomerases can be divided into several steps: 1) enzyme-DNA binding; 2) DNA cleavage, resulting in a covalent attachment between the protein and one terminus of the DNA nick; 3) DNA strand passage; 4) poststrand passage DNA religation concerted with the enzyme turnover (reviewed in Ref. UV photoproducts cause alterations of the DNA conformation that can affect the activity of DNA processin...

The present prognostic significance of HER2 is correlated to receptor expression level and points to the need to consider HER2 2+ and HER2 3+ tumors as distinct diseases with different outcomes and specific features.