It was previously reported that treatment with the sulfated polysaccharide fucoidan or the structurally similar dextran sulfate increased circulating mature white blood cells and hematopoietic progenitor/stem cells (HPCs) in mice and nonhuman primates; however, the mechanism mediating these effects was unclear. It is reported here that plasma concentrations of the highly potent chemoattractant stromal-derived factor 1 (SDF-1) increase rapidly and dramatically after treatment with fucoidan in monkeys and in mice, coinciding with decreased levels in bone marrow. In vitro and in vivo data suggest that the SDF-1 increase is due to its competitive displacement from heparan sulfate proteoglycans that sequester the chemokine on endothelial cell surfaces or extracellular matrix in bone marrow and other tissues. Although moderately increased levels of interleukin-8, MCP1, or MMP9 were also present after fucoidan treatment, studies in gene-ablated mice (GCSFR ؊/؊ , MCP1 ؊/؊ , or MMP9 ؊/؊ ) and the use of metalloprotease inhibitors do not support their involvement in the concurrent mobilization. Instead, SDF-1 increases, uniquely associated with sulfated glycan-mobilizing treatments and not with several other mobilizing agents tested, are likely responsible. To the authors' knowledge, this is the first published report of disrupting the SDF-1 gradient between bone marrow and peripheral blood through a physiologically relevant mechanism, resulting in mobilization with kinetics similar to other mobilizing CXC chemokines. The study further underscores the importance of the biological roles of carbohydrates. IntroductionStromal-derived factor 1 (SDF-1) is a highly potent chemoattractant both in vitro and in vivo for mature leukocytes and hematopoietic progenitor/stem cells (HPCs), which carry its receptor CXCR4. [1][2][3][4][5][6][7] This highly conserved chemokine is constitutively expressed by virtually all tissues, 8 including bone marrow (BM). 3 It is expressed as 2 alternatively spliced isoforms, the predominant ␣ form and the ␤ form containing 4 additional amino acids at the C terminus, each possessing a heparin-binding domain. 9,10 The SDF-1-CXCR4 interaction plays a dominant role in hematopoiesis, and mice deficient in either gene die in utero exhibiting defects in B-cell lymphopoiesis and BM myelopoiesis. 4,5 Additionally, a critical role for CXCR4 on human cells in engraftment to the BM of nonobese diabetic/severe combined immunodeficiency mice 6,7 has been shown. Although involvement of SDF-1 in mobilization-the egress of HPCs from the BM to the peripheral blood (PB)-has also been speculated, direct evidence has only recently been obtained in mice using a synthetic SDF-1 analog 11 or following injection with an adenovirus expressing human SDF-1. 12 Previously, we reported that the sulfated polysaccharide fucoidan (FucS) and the structurally similar dextran sulfate (DexS) can elevate circulating white blood cells (WBCs) and mobilize HPCs within hours in a selectin-independent manner in mice and nonhuman primates. 13...

Abstnset Treatment of human neutrophils with tumor necrosis factor-a (I'NF-u) resulted in an increase in concentration of ceramide and its catabolite, sphingosine. Sphingosine, a potent endogenous protein kinase C (PKC) inhibitor, as well as TNF-a, induced intemucleosomal DNA fragmentation and morphological changes characteristic of apoptotic cells. &amide and sphingosine-l-phosphate, the initial product of sphingosine catabolism. did not cause anontosis under our experimental conditions. In addition, 1-(5isoquinolinesulfonyl)-2-methylpiperazine (H7) and N, Ndimethylsphingosine (DMS): known as PKC inhibitors, also induced apoptosis, suggesting that-induction of apoptosis by sphingosine may be related to inhibition of PKC activity. These results indicate that sphingosine deacylated from ceramide may be an endogenous modulator mediating apoptotic signals by TNF-a in neutrophils.

In the study of apoptosis initiated by various signals including ligands binding to cell membrane receptors such as Fas and TNFRI, the sphingomyelin pathway and its resulting metabolites, the sphingolipids, have been suggested to be involved in the signaling pathway. In earlier studies we presented data which indicated that sphingosine (Sph) itself was increased during apoptosis induced by phorbol myristate acetate (PMA) in HL60 cells and tumor necrosis factor (TNF) in neutrophils, and when added exogenously was able to induce apoptosis. We report here that Sph and its methylated derivative N,N,-dimethylsphingosine (DMS) are able to induce apoptosis in cancer cells of both hematopoietic and carcinoma origin. In human leukemic cell lines CMK-7, HL60 and U937, treatment with 20 microM Sph for 6 hr caused apoptosis in up to 90% of cells. Human colonic carcinoma cells HT29, HRT18, MKN74 and COLO205 were shown to be more susceptible to apoptosis upon addition of DMS (>50%) than of Sph (<50%), yet were weakly or not sensitive to N,N,N-trimethylsphingosine (TMS). Under the same conditions, in the presence of serum, neither Sph-1-phosphate nor ceramide analogues C2-, C6- or C8-ceramide were able to induce apoptosis in any cell lines. However, in the absence of serum, ceramide analogues induced apoptosis in leukemia cell lines after 18 hr, yet much less so than Sph or DMS. Furthermore, apoptosis induced by Sph or DMS could not be inhibited by the ceramide synthase inhibitor fumonisin B1. Apoptosis was not induced by sphingolipids in primary culture cells, such as HUVEC or rat mesangial cells, but was apparent in transformed rat mesangial cells. Additionally, apoptosis induced by Sph, DMS or C2Cer was inhibited by protease inhibitors. Our data further support the evidence that the catabolic pathway of sphingomyelin involving Sph and other metabolites is an integral part of the apoptosis pathway.

Resistance to apoptosis plays an important role in tumors that are refractory to chemotherapy and ionizing radiation (IR). bax, which forms a heterodimer with bcl-2 and accelerates apoptosis, is not, or only weakly, expressed in most human breast cancer cells, and weak bax expression is considered to be related to the resistance of breast cancer cells to apoptosis. bax expression vector was introduced to human breast cancer MCF-7 cells, which exhibit weak expression of bax, to demonstrate its role of modulating radiation-induced apoptosis. bax overexpression in MCF-7 cells by stable transfection does not affect viability by itself, but each stable transfectant was more sensitive to IR than the parental MCF-7 cells. The degree of enhancement in radiosensitivity was dependent on the expression level of bax. IR upregulated p53 and p21WAF1 about 5- to 10-fold and downregulated bcl-2 and bcl-XL by 80-90% at 6 hr in both parent and bax stably transfected MCF-7 cells to the same degree. FACS analysis and DNA electrophoresis revealed that this sensitization was due to apoptosis. We suggest that exogenous bax expression might be one of the factors determining cellular radiosensitivity in MCF-7 breast cancer cells and may have therapeutic applications for enhancing radiation sensitivity in breast cancer cells.

Employing carbohydrate ligands, which have been extensively used to block selectin function in vitro and in vivo, we have examined the involvement of such ligands in stem͞progenitor cell mobilization in mice and monkeys. We found that sulfated fucans, branched and linear, are capable of increasing mature white cells in the periphery and mobilizing stem͞progenitor cells of all classes (up to 32-fold) within a few hours posttreatment in a dosedependent manner. To elicit the effect, the presence of sulfate groups was necessary, yet not sufficient, as certain sulfated hexosamines tested (chondroitin sulfates A or B) were ineffective. Significant mobilization of stem͞progenitor cells and leukocytosis was elicited in selectin-deficient mice (L ؊/؊ , PE ؊/؊ , or LPE ؊/؊ ) similar to that of wild-type controls, suggesting that the mode of action of sulfated fucans is not through blockade of known selectins. Other mechanisms have been entertained, in particular, the release of chemokines͞cytokines, including some previously implicated in mobilization. Significant increases were documented in the levels of seven circulating chemokines͞cytokines within a few hours after fucan sulfate treatment and support such a proposition. Additionally, an increase was noted in plasma metalloproteinase (MMP) 9, which might independently contribute to the mobilization process by enzymatically facilitating chemokine͞ cytokine release. Mobilization by sulfated polysaccharides provides a distinct paradigm in the mobilization process and uncovers an additional novel in vivo biological role for sulfated glycans. As similarly sulfated compounds were ineffective in vivo, the data also underscore the fact that polysaccharides with similar structures may elicit diverse in vivo effects.