FISH analysis of trisomy 8 and trisomy 18 in BLIN-4E and BLIN-4L Centromeres of chromosome 8, orange . Centromeres of chromosome 18, green . Chro- mosomes were counterstained with 4 Ј ,6-diamidino-2-phenylindole ( blue ). 

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... in the aforementioned immunodeficiency patients is We have discussed in detail the similar and distinct outcomes of IL-7 unknown. signaling on human and murine B-cell precursors (1). IL-7 is a One approach that may facilitate identification of BM stromal cell nonredundant BM-derived cytokine that is essential for murine B-cell products essential for the proliferation of human B-cell precursors is development (2). This criticality in murine IL-7 signaling is under- the use of in vitro human BM stromal cell cultures. We have devel- oped a culture system that uses freshly isolated nontransformed human BM stromal cells established from normal human BM (13, 14). A somewhat similar human BM stromal cell culture system has been used by Manabe et al. (15, 16) and Kumagai et al. (17) to study the apoptotic sensitivities of freshly isolated B-lineage ALL. We have also used this human BM stromal cell culture to establish a human pre-B ALL cell line that requires direct BM stromal cell contact for optimal survival/proliferation (18). The present report describes a new human B-lineage ALL cell line, designated BLIN-4, similarly established by plating freshly isolated leukemic cells on human BM stromal cells. We show that BLIN-4 cells respond to at least four distinct cytokines and unknown human BM stromal cell-derived molecule(s). We also describe sublines of BLIN-4 with differing sensitivities to survival/proliferation signals, suggesting that BLIN-4 may be a model for tumor progression in B-lineage ALL. Human B-cell development occurs as an ordered progression of maturation steps characterized by prominent checkpoints involving rearrangement and expression of heavy and light chain immunoglobulin genes (reviewed in 1). Although functional immunoglobulin rearrangements leading to expression of the pre-B-cell receptor and B-cell receptor are essential for successful development of B-lineage cells, traversal of these checkpoints is also influenced by the fetal liver and BM 3 microenvironment. The identity of the survival/proliferation factors that are essential for normal human B-cell development is only partially understood. We have discussed in detail the similar and distinct outcomes of IL-7 signaling on human and murine B-cell precursors (1). IL-7 is a nonredundant BM-derived cytokine that is essential for murine B-cell development (2). This criticality in murine IL-7 signaling is under- Establishment of BLIN-4. We have described previously a cell line (BLIN-2) that requires human BM stromal cells for optimal survival and proliferation (18). We now describe a new cell line, designated BLIN-4, that was also established by plating BM leukemic blasts from a patient with B-lineage ALL onto human BM stromal cells. Following an initial 1-month period of gradual leukemic cell death, BLIN-4 cells could be passaged as a stable cell line after ϳ 2 months in culture. Two sublines of the original parental BLIN-4 cell line were eventually derived designated BLIN-4E and BLIN-4L. The distinction between the two reflects the stochastic emergence of two sublines with different growth characteristics (see below) during the initial 3– 6 months in culture and is not meant to imply that BLIN-4L evolved directly from BLIN-4E. Fig. 1 shows that BLIN-4E and BLIN-4L express a qualitatively and quantitatively identical CD10 ϩ / CD19 ϩ /CD22 ϩ /CD40 ϩ B-lineage phenotype. The expression of cell surface VpreB on BLIN-4E cells in the absence of ␮ heavy chain is not without precedent in B-lineage ALL. Neither subline expressed CD7, CD20, CD34, CD80, or CD86. Interestingly, BLIN-4L cells expressed higher levels of CD49d/CD29 (VLA-4) compared with BLIN-4E cells, whereas both sublines expressed identical levels of CD11a/CD18 (LFA-1). Southern blot analysis with a human J H probe indicated that BLIN-4E and BLIN-4L sublines both harbored a single ϳ 8-kb rearrangement at the IgH locus with the other allele deleted (Fig. 2). Unfortunately, DNA was not available from the diagnostic BM specimen, so we are unable to characterize the IgH rearrangements in the original leukemic blasts. Cytogenetic analysis of the original BM specimen and the BLIN-4 sublines revealed multiple related clones that included 46,XX,r(4)/47,XX,r(4), ϩ 18/47,XX,r(4), ϩ 8/and 48,XX,r(4), ϩ 8, ϩ 18. Because of the low yield of mitotic cells in the original BM aspirate and early passages of BLIN-4, we were unable to accurately estimate the relative frequencies of the karyotypically distinct subclones by G-banding analysis. Therefore, we performed FISH using chromosome 8- and 18-specific probes. Analysis of 200 interphase cells in the original BM specimen revealed that trisomy 8 was present in 11.5% of cells, and trisomy 18 was present in 2.5% of cells. Analysis of 300 interphase BLIN-4E cells revealed that 89% of the cells had a trisomy 8, and 0% had a trisomy 18. In marked contrast, analysis of 300 interphase BLIN-4L cells indicated that 88% had trisomy 18, and 1% had a trisomy 8. Fig. 3 shows representative FISH profiles of the two sublines. BM Stromal Cell Dependency of BLIN-4 Sublines. Approximately 10 months following initial plating, BLIN-4 cells slowly proliferated on human BM stromal cells and had a doubling time of ϳ 10 days (Fig. 4). BLIN-4 cells slowly died in the absence of BM stromal cells, and IL-7 marginally delayed cell death. Addition of IL-7 at day 0 significantly enhanced ( P ϭ 0.001) proliferation of BLIN-4 cells on BM stromal cells between days 7 and 15 (Fig. 4). Substitution of FLT3-L for IL-7 gave similar results (data not shown). Direct contact with BM stromal cells was necessary for optimal proliferation, because only ϳ 25% of contact-dependent proliferation remained when BLIN-4 cells were incubated in Transwell inserts above the BM stromal cell monolayer (data not shown). Approximately 10 months after initiating the BLIN-4 cell line, we noticed that the cells were exhibiting enhanced survival in X-VIVO 10 serum-free medium alone. We designated this subline BLIN-4L to distinguish it from the early passaged subline (BLIN-4E) with more stringent culture requirements. Table 1 shows the results of two experiments comparing the survival/proliferation characteristics of BLIN-4E and BLIN-4L. In these experiments skin fibroblasts were used in lieu of BM stromal cells as an adherent cell monolayer. Consistent with the results shown in Fig. 4, BLIN-4E cells were Ͼ 90% dead by day 7 in the absence of cytokines and/or fibroblasts. Inclusion of IL-7 and FLT3-L only marginally inhibited cell death. BLIN-4E cells essentially survived for 2 weeks when cultured on skin fibroblasts. Addition of IL-7 and FLT3-L to the fibroblasts led to a significant enhancement in BLIN-4E cell number compared with BLIN-4E cells maintained on fibroblasts alone ( P ϭ Ͻ 0.001). However, in the presence of exogenous IL-7/FLT3-L and skin fibroblasts, BLIN-4E cells only expanded 4- to 5-fold by day 14 (Table 1). In marked contrast, BLIN-4L cells proliferated in medium alone, and proliferation was comparably enhanced by culturing in either IL-7/ FLT3-L or on skin fibroblasts. Maximum expansion occurred when BLIN-4L cells were cultured in the presence of IL-7/FLT3-L and skin fibroblasts, with 90- to 260-fold increases in cell number by day 14. These collective results indicate that BLIN-4L cells are dramatically more sensitive to proliferation signals transduced by cytokines and fibroblast adherent layers than are BLIN-4E cells. As noted above, BLIN-4L expressed higher levels of VLA-4 than BLIN-4E (Fig. 1). Consistent with this quantitative difference, BLIN-4L cells were approximately twice as adherent to BM stromal cells as BLIN-4E cells when measured in static (nonfluid shear) adhesion assays (data not shown). However, the adherence of BLIN-4L cells to BM stromal cells was completely blocked by a mAb to the CD29/ 1 subunit, whereas the adherence of BLIN-4E cells was not. Cytokine Responsiveness of BLIN-4L. The capacity of BM stromal cells to support the proliferation of BLIN-4L cells could be partially substituted by conditioned medium from BM stromal cells (data not shown). We therefore screened a panel of cytokines shown previously to stimulate human or murine B-cell precursors in other experimental systems. The panel included: IL-3, IL-6, IL-11, bFGF, FLT3-L, IGF-I, OSM, SCF, and SDF-1 ␣ . As expected, IL-7 enhanced proliferation of BLIN-4L cells (Fig. 5). The pool of cytokines (rep- resented by IL-7 ϩ IL-3, IL-6, IL-11, bFGF, FLT3-L, IGF-I, OSM, SCF, and SDF-1 ␣ ) promoted an additional 3-fold increase over IL-7 alone. When individual components of the cytokine panel were tested for their capacity to cooperate with IL-7, FLT3-L was the only cytokine that promoted the proliferation of BLIN-4L cells (Fig. 5). Additional results in Fig. 6 demonstrated that BLIN-4L cells exhibited a dose-dependent response to FLT3-L and IL-7. Note that the data in Fig. 6 represent survival/proliferation at day 7 only. Thus, inclusion of 10 ng/ml IL-7 resulted in a 63% increase in BLIN-4L cell number at day 7 (compared with day 0 input), and inclusion of 10 ng/ml FLT3-L resulted in a 31% increase in BLIN-4L cell number at day 7 (compared with day 0 input). However, inclusion of 10 ng/ml of both cytokines led to a synergistic 352% increase in BLIN-4L cell number at day 7 (compared with day 0 input). We have reported previously (14) that human BM stromal cells, which constitute the supportive microenvironment in our culture system, produce extremely small amounts of IL-7 (1.0 –2.0 pg/ml). However, this quantity of IL-7 (in whatever physical disposition it adopts in our BM stromal cell culture system) has no bioactivity for normal B-cell precursors (14). FLT3-L has been reported to be produced by human BM stromal cells (25). We confirmed that report by finding that fetal BM stromal cells used in the current study produced ϳ 10 pg/ml of FLT3-L as measured by ELISA (data not shown). We therefore examined whether the ...