In clinical trials, the tyrosine kinase inhibitor STI571 has proven highly effective in reducing leukemic cell burden in chronic myeloid leukemia (CML). The overall sensitivity of CML CD34 ؉ progenitor cells to STI571 and the degree to which cell death was dependent on cell cycle status were determined. Stem cells (Lin ؊ CD34 ؉ ) from the peripheral blood of patients with CML in chronic phase and from granulocyte-colony-stimulating factor-mobilized healthy donors were labeled with carboxy-fluorescein diacetate succinimidyl diester dye to enable highresolution tracking of cell division. Then they were cultured for 3 days with and without growth factors ؎ STI571. After culture, the cells were separated by fluorescence-activated cell sorting into populations of viable quiescent versus cycling cells for genotyping. For healthy controls, in the presence of growth factors, STI571 affected neither cell cycle kinetics nor recovery of viable cells. In the absence of growth factors, normal cells were unable to divide. For CML samples, in the presence or absence of growth factors, the response to STI571 was variable. In the most sensitive cases, STI571 killed almost all dividing cells; however, a significant population of viable CD34 ؉ cells was recovered in the undivided peak and confirmed to be part of the leukemic clone. STI571 also appeared to exhibit antiproliferative activity on the quiescent population. These studies confirm that CML stem cells remain viable in a quiescent state even in the presence of growth factors and STI571. Despite dramatic short-term responses in vivo, such in vitro insensitivity to STI571, in combination with its demonstrated antiproliferative activity, could translate into disease relapse after prolonged therapy. IntroductionChronic myeloid leukemia (CML) is a clonal myeloproliferative disease characterized by the t(9;22) chromosome translocation that, in turn, creates the BCR-ABL oncogene. [1][2][3] The fusion gene product is a p210 oncoprotein containing a constitutively active tyrosine kinase that confers certain growth advantages to the Philadelphia-positive (Ph ϩ ) clone compared with normal hematopoietic cells. 4 We have demonstrated recently the existence of a population of rare, primitive, quiescent stem cells in all chronic-phase CML patient samples, whether derived from peripheral blood or bone marrow. These stem cells are predominantly Ph ϩ , express high levels of CD34 ϩ but lack the markers CD38, CD45RA, or CD71, and can spontaneously exit G 0 to enter a continuously proliferating state, either in vitro or to produce Ph ϩ progeny in immunocompromised mice in vivo. 5,6 Many cancers are treated with relatively nonselective cytotoxic drugs that affect normal and malignant cells. Because most available chemotherapeutic agents show some degree of S-phase specificity, cells that are not actively dividing may prove resistant to such drugs. This raises the possibility that the quiescent leukemic cells we have identified in patients with CML are likely to survive standard chemotherapy r...
Ultrasound-induced bubble activity (cavitation) has been recently shown to actively transport and improve the distribution of therapeutic agents in tumors. However, existing cavitation-promoting agents are micron-sized and cannot sustain cavitation activity over prolonged time periods because they are rapidly destroyed upon ultrasound exposure. A novel ultrasound-responsive single-cavity polymeric nanoparticle (nanocup) capable of trapping and stabilizing gas against dissolution in the bloodstream is reported. Upon ultrasound exposure at frequencies and intensities achievable with existing diagnostic and therapeutic systems, nanocups initiate and sustain readily detectable cavitation activity for at least four times longer than existing microbubble constructs in an in vivo tumor model. As a proof-of-concept of their ability to enhance the delivery of unmodified therapeutics, intravenously injected nanocups are also found to improve the distribution of a freely circulating IgG mouse antibody when the tumor is exposed to ultrasound. Quantification of the delivery distance and concentration of both the nanocups and coadministered model therapeutic in an in vitro flow phantom shows that the ultrasound-propelled nanocups travel further than the model therapeutic, which is itself delivered to hundreds of microns from the vessel wall. Thus nanocups offer considerable potential for enhanced drug delivery and treatment monitoring in oncological and other biomedical applications.
Purification of hepatitis C virus (HCV) from sera of infected patients has proven elusive, hampering efforts to perform structure-function analysis of the viral components. Recombinant forms of the viral glycoproteins have been used instead for functional studies, but uncertainty exists as to whether they closely mimic the virion proteins. Here, we used HCV virus-like particles (VLPs) generated in insect cells infected with a recombinant baculovirus expressing viral structural proteins. Electron microscopic analysis revealed a population of pleomorphic VLPs that were at least partially enveloped with bilayer membranes and had viral glycoprotein spikes protruding from the surface. Immunogold labeling using specific monoclonal antibodies (MAbs) demonstrated these protrusions to be the E1 and E2 glycoproteins. A panel of anti-E2 MAbs was used to probe the surface topology of E2 on the VLPs and to compare the antigenicity of the VLPs with that of truncated E2 (E2 660 ) or the full-length (FL) E1E2 complex expressed in mammalian cells. While most MAbs bound to all forms of antigen, a number of others showed striking differences in their abilities to recognize the various E2 forms. All MAbs directed against hypervariable region 1 (HVR-1) recognized both native and denatured E2 660 with comparable affinities, but most bound either weakly or not at all to the FL E1E2 complex or to VLPs. HVR-1 on VLPs was accessible to these MAbs only after denaturation. Importantly, a subset of MAbs specific for amino acids 464 to 475 and 524 to 535 recognized E2 660 but not VLPs or FL E1E2 complex. The antigenic differences between E2 660, FL E1E2, and VLPs strongly point to the existence of structural differences, which may have functional relevance. Trypsin treatment of VLPs removed the N-terminal part of E2, resulting in a 42-kDa fragment. In the presence of detergent, this was further reduced to a trypsin-resistant 25-kDa fragment, which could be useful for structural studies.Hepatitis C virus (HCV), the major cause of non-A, non-B hepatitis, is an enveloped virus classified in the Flaviviridae family (31, 52). The positive-strand viral RNA genome encodes a single polyprotein of approximately 3,010 amino acids that is processed into functional proteins by host and viral proteases (52, 53). The putative HCV structural proteins comprising the core and the two envelope glycoproteins E1 and E2 are located within the N terminus of the polyprotein, while the nonstructural proteins reside within the C-terminal part. Analysis of the structural features of the HCV virion has been hampered by the inability to propagate the virus in vitro. In addition, it has been difficult to purify sufficient quantities of virus from infected patient plasma or tissue. By analogy with other flaviviruses, the core is presumed to be enveloped in a lipid bilayer containing the viral glycoproteins. Glycoproteins E1 and E2 are believed to be type I integral transmembrane proteins, with C-terminal hydrophobic anchor domains (reviewed in reference 40). In vitro exp...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.