Search

High-dose chemotherapy (HDC) with hematopoietic support appears promising in the treatment of breast cancer, although reinfusion of contaminating tumor cells may contribute to disease relapse. Ex vivo expansion may reduce tumor cell content through use of a small inoculum volume and by passive purging during culture. We assessed the ex vivo expansion potential of tumor cell positive bone marrow (BM) from breast cancer patients and the effect of ex vivo expansion on tumor cell content. Cryopreserved/thawed mononuclear cell (C/T MNC) BM harvests with known tumor cell contamination (n = 7) were assessed for tumor cells pre- and post-expansion using immunocytochemical (ICC) staining. Pre-expansion inoculum samples contained a range of 6–2128 tumor cells per 5.0 × 106 nucleated cells. Ex vivo expansion resulted in fold expansions of 6.67 and 11.37 for total cells and CFU-GM, respectively. Tumor cells were undetectable in four of the seven post-expansion samples and were reduced in the remaining three samples. The data demonstrate passive purging of breast cancer cells during ex vivo expansion, with hematopoietic progenitor cell expansion comparable to that of normal BM. Reduction in tumor cell number contained in the small volume culture inoculum combined with passive purging during the ex vivo expansion process suggest a potential 2–4+ log reduction in tumor cell content in the reinfused cell product. [ABSTRACT FROM AUTHOR]

Adhesion of normal colony-forming cells (CFC) to bone marrow (BM) stroma and the extracellular matrix (ECM) component fibronectin (FN) depends at least in part on the alpha4beta1 and alpha5beta1 integrins and the CD44 receptor. Aside from anchoring progenitors in the marrow microenvironment, beta1 integrin-dependent adhesion of normal CFC is associated with inhibition of their proliferation. In contrast to normal CFC, chronic myelogenous leukemia (CML) Ph+ CFC adhere significantly less to either stroma or FN. CML Ph+ CFC proliferation is also not inhibited by coculture with stroma or FN. However, equal numbers of alpha4, alpha5, and beta1 integrins and CD44 are present on CML and normal CD34+ cells. We have previously demonstrated that beta1-dependent adhesion to and subsequent proliferation inhibition by FN can be restored when CML Ph+ CFC are incubated with the beta1 integrin activating antibody, 8A2, and demonstrated a role for the alpha5beta1 integrin in this phenomenon. Since the integrin alpha4beta1 and the proteoglycan form of CD44 may cooperate in establishing normal CFC adhesion to FN, we examined if treatment of CML Ph+ CFC with 8A2 also restores the cooperativity between beta1 integrins and CD44. We demonstrate that 8A2 induces adhesion of CML Ph+ CFC not only to intact FN but also to alpha4beta1, alpha5beta1, and proteoglycan binding fragments of FN. 8A2-induced adhesion to these fragments and peptides also results in a significant inhibition of the proliferation of CML Ph+ CFC. Addition of antibodies to either the alpha5, alpha4, or beta1 integrins, antibodies against the CD44 receptor, or removal of chondroitin sulfate glycosaminoglycans from the surface of CML CD34+ HLA-DR+ cells significantly reduced the 8A2-induced adhesion to and adhesion-mediated inhibition of proliferation by FN. These studies demonstrate that activation of beta1 integrins on CML Ph+ CFC not only results in upregulation of beta1 integrin-dependent adhesion and adhesion-mediated inhibition of proliferation, but also in the restoration of cooperation between beta1 integrins and CD44. These studies suggest that decreased beta1 integrin avidity may also affect the function of the proteoglycan adhesion receptor CD44, both of which may contribute to the abnormal circulation and expansion of malignant progenitors in CML. [ABSTRACT FROM AUTHOR]

Adhesion of normal colony-forming cells (CFC) to bone marrow (BM) stroma and the extracellular matrix (ECM) component fibronectin (FN) depends at least in part on the α4β1 and α5β1 integrins and the CD44 receptor. Aside from anchoring progenitors in the marrow microenvironment, β1 integrin-dependent adhesion of normal CFC is associated with inhibition of their proliferation. In contrast to normal CFC, chronic myelogenous leukemia (CML) Ph+ CFC adhere significantly less to either stroma or FN. CML Ph+ CFC proliferation is also not inhibited by coculture with stroma or FN. However, equal numbers of α4, α5, and β1 integrins and CD44 are present on CML and normal CD34[SUP+] cells. We have previously demonstrated that β1-dependent adhesion to and subsequent proliferation inhibition by FN can be restored when CML Ph+ CFC are incubated with the β1 integrin activating antibody, 8A2, and demonstrated a role for the α5β1 integrin in this phenomenon. Since the integrin α4β1 and the proteoglycan form of CD44 may cooperate in establishing normal CFC adhesion to FN, we examined if treatment of CML Ph+ CFC with 8A2 also restores the cooperativity between β1 integrins and CD44. We demonstrate that 8A2 induces adhesion of CML Ph+ CFC not only to intact FN, but also to α4β1, α5β1, and proteoglycan binding fragments of FN. 8A2-induced adhesion to these fragments and peptides also results in a significant inhibition of the proliferation of CML Ph+ CFC. Addition of antibodies to either the α5, α4, or β1 integrins, antibodies against the CD44 receptor, or removal of chondroitin sulfate glycosaminoglycans from the surface of CML CD34[SUP+] HLA-DR[SUP+] cells significantly reduced the 8A2-induced adhesion to and adhesion-mediated inhibition of proliferation by FN. These studies demonstrate that activation of β1 integrins on CML Ph+ CFC not only results in... [ABSTRACT FROM AUTHOR]

Ex vivo expansion of bone marrow (BM) mononuclear cells (MNC) in a perfused culture system produces stem-progenitor cell type(s) in sufficient number(s) for hematopoietic reconstitution. The limitations in using fresh BM MNC for ex vivo expansion include additional cell processing and inflexibility in patient treatment. Cryopreservation of whole bone marrow (WBM) eliminates processing costs of MNC or CD34+ cell selection and allows for flexibility in patient treatment. We developed a convenient system to cryopreserve and thaw small volume WBM aspirations (n = 13) and then compared the expandability of unprocessed normal cryopreserved/thawed (C/T) WBM to that of fresh BM MNC cultured in the presence of erythropoietin, PIXY 321, and Flt3-ligand. Ex vivo expansion potential was retained in WBM aspirates after C/T. When initiated with 225 million viable nucleated cells, clinical scale expansion cultures (n = 6) yielded 9.7+/-2.8 x 10(8) total cells, which contained 10.4+/-5.8 x 10(6) colony-forming units-granulocyte-macrophage (CFU-GM), 1.3+/-1.4 x 10(4) LTCIC, and 2.2 x 10(6) CD34+Lin- cells, sufficient cell numbers for clinical use. These studies demonstrate that ex vivo perfusion culture expansion of unfractionated C/T WBM (< or =30 ml) provides doses of stem-progenitor cells similar in composition to expanded fresh BM MNC, previously demonstrated to achieve hematopoietic reconstitution.

Hematopoiesis takes place in close contact with the marrow microenvironment. Normal progenitors adhere through a variety of receptors to stroma and extracellular matrix components, including fibronectin. Adhesion through integrins to fibronectin may not only serve to anchor progenitors to the microenvironment but also to directly alter the proliferative behavior of normal hematopoietic progenitors. Chronic myelogenous leukemia (CML) is a malignant disease of the hematopoietic stem cell. At the molecular level, CML is characterized by the BCR/ABL gene rearrangement which encodes for the oncoprotein, p210bcr-abl. Presence of the p210bcr-abl tyrosine kinase is necessary and sufficient for the malignant transformation of hematopoietic cells. Clinically, CML is characterized by an abnormal, premature release of primitive progenitors and precursors in the blood and by the continuous proliferation of the malignant progenitor population. In vitro, CML progenitors fail to adhere to or be regulated by marrow stroma. Since CML progenitors express similar numbers of integrin adhesion receptors as normal progenitors, functional rather than quantitative differences of these receptors on CML progenitors may be responsible for the abnormal circulation and proliferation of the malignant clone. In this manuscript we will review the role of integrin adhesion receptors present on normal hematopoietic progenitors in the regulation of their proliferation and discuss signal transduction mechanisms that may be responsible for these effects. We will also discuss the integrin defect in CML which may be caused by the presence of the oncoprotein, P210bcr-abl, and may explain the abnormal trafficking and proliferation observed in CML.

Chronic myelogenous leukemia (CML) is a malignant disease of the hematopoietic stem cell characterized by abnormal circulation and proliferation of malignant progenitors. In contrast to their normal counterparts, CML progenitors adhere poorly to bone marrow stroma or fibronectin (FN). Aside from anchoring progenitors in the marrow microenvironment, beta1 integrin-dependent adhesion of normal progenitors is also associated with inhibition of their proliferation. As the beta1 integrin expression on CML progenitors is normal, we hypothesized that decreased integrin affinity may underlie the abnormal adhesive and proliferative characteristics of CML progenitors. We examined the effect of affinity modulation by the activating antibody 8A2 on the adhesion and proliferation of CML progenitors and the CML cell line, K562. 8A2 induced alpha5Beta1-dependent adhesion of Philadelphia chromosome-positive (Ph+) CD34+/HLA-DR+ cells and K562 cells to FN. Increased adhesion was 8A2- and FN concentration-dependent, time-dependent, and energy-dependent. Further, 8A2-induced adhesion to FN significantly inhibited the proliferation of malignant CML progenitors as well as K562 cells independent of cell differentiation, necrosis, or apoptosis. These studies demonstrate that affinity modulation of the alpha5Beta1 integrin on CML progenitors and K562 cells by 8A2 results in increased adhesion to FN with subsequent decreased proliferation, suggesting that decreased beta1 integrin affinity contributes to the abnormal circulation and proliferation of malignant progenitors in CML.

The specific activity of galactose-1-phosphate uridyltransferase was measured in fetal and neonatal guinea pig liver during the first 10 days of age. Activity was approximately six times greater than in adult animals, and peaked during the first 48 h after birth. Activity dropped sharply during the next 2 days, followed by a gradual decline. Liver galactose and glycogen levels were stable throughout the study period. Liver glucose rose significantly after birth, then dropped slightly. The pattern of uridyltransferase in the guinea pig is similar to that reported for phosphoenolpyruvate carboxylkinase and pyruvate carboxylase. Compared to the rat, specific activity of uridyltransferase peaked earlier, possibly due to the guinea pig's advanced maturity.