B cell chronic lymphocytic leukemia (B-CLL) consists of the accumulation of malignant cells that apparently escape normal apoptotic regulation. We have studied the role of ␣4␤1 integrin/fibronectin interaction in preventing apoptosis of these cells in vitro. B cells from 16 patients showed constant expression of ␣4␤1 and little or no ␣5␤1. B-CLL cells cultured on fibronectin or two previously described fibronectin recombinant fragments (H89 and H0) which contain the ligands for ␣4␤1, consistently showed higher viability than control cells cultured on poly-lysine. The H89 fragment, containing the high affinity ligand CS-1, was the most efficient substrate with mean cell viability values of 72, 60 and 35% at days 2, 5 and 8 of culture, respectively. For control cells these values were 40, 27 and 15%, respectively. Parallel cell cycle analysis confirmed these results. The anti-apoptotic effect required direct contact with immobilized substrata since it was not observed when using B-CLL conditioned media alone or when clustering ␣4␤1 with specific mAbs in suspension. Quantitation of the apoptosis regulatory proteins Bcl-2 and Bax revealed that cells cultured on the H89 fragment showed high/moderate levels of Bcl-2 (with some interpatient variation) and low levels of Bax resulting in an elevated Bcl-2/Bax ratio. These results indicate that adhesion of B-CLL cells to fibronectin upregulate the Bcl-2/Bax ratio and this may contribute to the anti-apoptotic effect induced via ␣4␤1 integrin.

The region of fibronectin encompassing type III repeats 4 -6 contains a low affinity heparin binding domain, but its physiological significance is not clear. We have studied whether this domain is able to interact Fibronectin (Fn) 1 is an extracellular matrix and plasma protein composed of structural domains that contain binding sites for other macromolecules (fibrin, heparin/proteoglycans, collagen), as well as for cells (reviewed in Hynes (1)). The NH 2 -terminal heparin binding domain or Hep I (see Fig. 1) also interacts with cell surfaces by binding to uncharacterized molecules and is mainly involved in formation of Fn matrices (1, 2). The Hep II domain displays the highest avidity for heparin and contains specific sequences which bind proteoglycans and/or the ␣4␤1 integrin at the cell surface and induce cell adhesion (3-5). One of these sites is H1, which is a ligand for ␣4␤1 in melanoma (6) and lymphoid cells (7). Two other sequences, CS-1 and CS-5, located within the IIICS segment (outside the Hep II domain) are also ligands for ␣4␤1 (8 -11) and bind with different affinities (12). These previous studies have established that ␣4␤1 may bind several sequences in the COOHterminal region of Fn and that these interactions are particularly important in lymphoid cells, where ␣4␤1 is highly expressed.The central Hep III domain is less well studied because it binds heparin only at low salt concentration, and the physiological significance of this interaction is unclear. Using DNA affinity chromatography, we and others previously isolated proteolytic fragments from this region of Fn, which displayed low affinity binding to heparin. These include a 14-kDa fragment corresponding to FN-III1 repeat (13) and two fragments of 18 -20 kDa (FN-III4-1/2 5 repeats) (14) and 30 kDa (FN-III4 -6 repeats) (15) from human and bovine plasma Fn, respectively. We also previously reported that an 80-kDa tryptic fragment (FN-III4-1/2 FN-III11) binds heparin with low avidity (16). It is not known whether this heparin binding domain also interacts with cells.The present study was undertaken to further characterize the properties of the Hep III domain of Fn. To this effect, we have prepared recombinant fragments encompassing type III homology repeats from this region and have studied their cell binding activities. We show that a fragment containing the FN-III5 repeat mediates adhesion of T and B lymphoid cells efficiently. Furthermore, we have identified a novel 6-amino acid-sequence as the active site in FN-III5, and we show that activated ␣4␤1 and ␣4␤7 integrins are receptors for this sequence and the FN-III5 fragment. These results therefore establish a novel function for the Hep III Fn domain.

The molecular pathogenesis of B cell chronic lymphocytic leukemia (B-CLL), the most common form of leukemia, remains unknown. We have used the mRNA differential display technique to analyze genes that may be involved in the development/progression of B-CLL. We have identified the tumor suppressor retinoic acid receptor responder 3 (RARRES3) as a B-CLL-related gene. RARRES3 maps to chromosome band 11q23, a region frequently deleted in lymphoproliferative disorders. To assess the potential involvement of RARRES3 in leukemogenesis, we examined 24 cases of B-CLL, 10 of acute lymphocytic leukemia (ALL) and five related cell lines by RT-PCR and sequence analyses. We report a correlation between RARRES3 down-regulation and B-CLL progression. We also found decreased RARRES3 gene levels in ALL cases and in the five cell lines studied. We did not find mutations in any of the leukemia samples assayed, including those with 11q23 deletion. These results indicate that RARRES3 may play a role in B-CLL progression. Leukemia (2001) 15, 1521-1526.

Malignant cell accumulation in B-cell chronic lymphocytic leukemia (B-CLL) is primarily caused by defective apoptosis rather than increased proliferation. To further understand the role of Bcl-2 family members, known regulators of apoptosis, in the abnormal B-CLL survival, we have measured their mRNA levels in fresh B-CLL cells and in cultures undergoing spontaneous apoptosis. Using RNA protection assays we found constitutive expression of most bcl-2 members with high levels of bcl2, bcl-w, bad, bak, bax, and the bcl-2/bax ratio, compared to normal PBL. Spontaneous apoptosis of B-CLL cells by in vitro culture resulted in decreased bcl-2, bcl-w, bfl-1, mcl-1, bak, bax, and bcl-2/bax expression. The pro-apoptotic member bik was only expressed in 5/19 cases and was not modulated during apoptosis, suggesting that bik is not involved in this process. Thus, several Bcl-2 family genes are regulated during B-CLL spontaneous apoptosis and their relative levels may contribute to in vivo progression of the disease.

Apoptosis is a regulated event crucial to the development and proliferation of normal and malignant B cells. We have studied the role of signals delivered via alpha4 integrin on apoptosis triggered by three different pathways on these cells. For apoptosis induced by serum deprivation, culturing B cells on the recombinant fibronectin fragment H89, a known ligand for alpha4beta1 integrin, resulted in statistically significant (P < 0.005) higher viability values (68%, 65% and 67%) for Ramos, Nalm-6 and EHEB cells, respectively, than culturing cells on poly lysine (42%, 42% and 48%). An antialpha4 MoAb reverted the protecting effect, thus confirming that it was due specifically to alpha4 engagement. Similarly, cells cultured on FN-III4-5, a recently identified fibronectin region which binds activated alpha4 integrin, also showed statistically significant higher viability than poly lysine cultures. Alpha4 engagement however, did not prevent apoptosis induced on Ramos cells via surface IgM. Adhesion of IM-9 cells, a myeloma cell line carrying functional Fas receptors, to the H89 fragment neither increased cell viability upon triggering apoptosis via Fas when compared to poly lysine. These results indicate that alpha4 signalling may overcome B cell apoptosis induced by the lack of growth factors but does not seem to affect the IgM or Fas apoptotic pathways, thus suggesting different intracellular mechanisms for these processes.