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Originally published online as doi:10.1189/jlb.0607425 on September 7, 2007

Published online before print September 7, 2007
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(Journal of Leukocyte Biology. 2007;82:1363-1364.)
© 2007 by Society for Leukocyte Biology

The antiapoptotic protein Api5 and its partner, high molecular weight FGF2, are up-regulated in B cell chronic lymphoid leukemia

Pavel Krejci*,{dagger},1, Katerina Pejchalova{ddagger}, Barry E. Rosenbloom§, Fred P. Rosenfelt§, Elizabeth L. Tran§, Henrik Laurell|| and William R. Wilcox{ddagger},¶

* Department of Animal Physiology and Immunology, Masaryk University, Czech Republic;
{dagger} Department of Cytokinetics, Institute of Biophysics ASCR, Czech Republic;
{ddagger} Medical Genetics Institute, Cedars-Sinai Medical Center, Los Angeles, California, USA;
§ Tower Hematology Oncology, Beverly Hills, California, USA;
|| INSERM U858, Toulouse, France; and
Department of Pediatrics, University of California, Los Angeles, School of Medicine, Los Angeles, California, USA

1Correspondence: Department of Animal Physiology and Immunology, Masaryk University, Kotlarska 2, 61137 Brno, Czech Republic. E-mail: krejcip{at}sci.muni.cz

Key Words: AAC-11 • FIF

The apoptosis inhibitor-5 [Api5; antiapoptosis clone 11 (AAC-11); fibroblast growth factor-2 (FGF2)-interacting factor (FIF)] is a 504-aa nuclear protein frequently up-regulated in tumor cells. Several studies have shown potent antiapoptotic action of Api5, which appears to be, at least in part, mediated via suppression of apoptosis depending on the E2F family of transcription factors [1 2 3 4 ].

Human FGF2 is a prototypic member of the FGF family of growth factors and exists in several variants generated by alternative translation of a single transcript. The smallest variant, 18 kDa FGF2, is released from cells and acts through binding and activation of cell-surface FGF-receptor tyrosine kinases. In contrast, the high molecular weight (HMW) FGF2 variants (22, 22.5, and 24 kDa) localize predominantly to the nucleus and have an intracrine, FGF receptor-independent mode of action [5 ]. It is interesting that HMW but not 18 kDa FGF2 interacts with Api5 in mammalian cells [3 ]. Similar to Api5, HMW FGF2 has a prosurvival action in various experimental models [6 ], although it is unknown to what extent HMW FGF2 and Api5 depend functionally on each other.

In B cell chronic lymphoid leukemia (B-CLL), FGF2 is up-regulated with an 18-kDa FGF2 peripheral blood concentration significantly exceeding control levels in the majority of patients with advanced disease [7 ]. This deregulation also involves HMW FGF2, which is overexpressed in B-CLL cells [8 ]. As Api5 represents the binding partner of HMW FGF2 [3 ], we asked whether Api5 is also up-regulated in B-CLL. We compared the expression of Api5 and HMW FGF2 in PBMCs obtained from eight B-CLL patients and five control individuals. Figure 1A shows up-regulation of 55 kDa (full-length) Api5 and HMW FGF2 in the majority of B-CLL patients when compared with control samples. In addition to the full-length Api5, a 25-kDa truncated variant of Api5, generated by alternative translation [1 ], was found in some samples.


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  		Figure 1. Api5 and FGF2 expression in B-CLL. (A) PBMCs were isolated from five controls and eight B-CLL patients via density centrifugation (Histopaque-1077, Sigma Chemical Co., St. Louis, MO, USA), and 15 µg extracted protein was subjected to Western blotting (WB) with FGF2 (Sigma Chemical Co.) and Api5 (a gift from Raphael Rubin, Jefferson Medical College, Philadelphia, PA, USA) antibodies. B-CLL stage (Rai) patients’ age/sex and FGF2 isoforms are indicated. Note the up-regulation of full-length Api5 (55 kDa) and FGF2 in B-CLL. The levels of a truncated Api5 variant (25 kDa) are also shown. WB with actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) serves as loading control. (B) PBMCs were isolated from six controls and five B-CLL patients, and 11 µg extracted protein was subjected to FGF2, Api5, and actin WB (upper panel). The same PBMCs were enriched for B cells using the negative B cell isolation kit (Dynal Biotech, Oslo, Norway), according to the manufacturer’s protocol (lower panel; 6 µg total protein loaded). (C) PBMCs isolated from three controls and one B-CLL patient were subjected to B cell enrichment via positive selection (CD19 Dynabeads, Dynal Biotech), followed by antibody detachment (DETACHaBEAD CD19, Dynal Biotech), and 2 µg extracted protein was analyzed for Api5, FGF2, and actin by WB. The percentage of B cell enrichment, determined by staining with PE-conjugated CD19 antibody (BD PharMingen, San Jose, CA, USA), followed by flow cytometry, is indicated. Note the lower expression of 25 kDa Api5 in the samples enriched for B cells when compared with PBMCs, suggesting it comes from a cellular source other than B cells. Also note that control 61/F (blots A–C) and B-CLL 71/M (blots B and C) represent the same individuals.

 
The up-regulation of full-length Api5 in B-CLL (Fig. 1A) might be specific to the leukemic clones or simply represent B cell enrichment in the B-CLL PBMCs compared with control PBMCs. We therefore determined the levels of Api5 and FGF2 in PBMCs enriched for B cells via negative selection, i.e., removal of T cells, NK cells, and monocytes using magnetic bead separation. Compared with controls, there was a clear up-regulation of full-length Api5 and HMW FGF2 in the B cell-enriched PBMCs of all five B-CLL individuals (Fig. 1B) . Similar results were obtained with CD19+ B cells purified from PBMCs via magnetic bead separation, thus confirming that B-CLL leukemic clones have up-regulated full-length Api5 and HMW FGF2 (Fig. 1C) . Considering the nature of the defect leading to B-CLL, i.e., decreased apoptosis of mature B cells, Api5 and HMW FGF2 may contribute to the pathology of B-CLL.

ACKNOWLEDGEMENTS

This study was supported by the Winnick Family Scholars Award (W. R. W.), Ministry of Education, Youth and Sports of the Czech Republic (MSM0021622430), and Cedars-Sinai Medical Genetics Institute fellowship (P. K.). We thank Dr. R. Rubin for the AAC-11 antibody, P. Lin for assistance with FACS analyses, and to patients and control individuals for participation in the study.

Received June 22, 2007; revised June 22, 2007; accepted July 9, 2007.

REFERENCES

  1. Tewari, M., Yu, M., Ross, B., Dean, C., Giordano, A., Rubin, R. (1997) AAC-11, a novel cDNA that inhibits apoptosis after growth factor withdrawal Cancer Res. 57,4063-4069[Abstract/Free Full Text]
  2. Kim, J. W., Cho, H. S., Kim, J. H., Hur, S. Y., Kim, T. E., Lee, J. M., Kim, I. K., Namkoong, S. E. (2000) AAC-11 overexpression induces invasion and protects cervical cancer cells from apoptosis Lab. Invest. 80,587-594[Medline]
  3. Van Den Berghe, L., Laurell, H., Huez, I., Zanibellato, C., Prats, H., Bugler, B. (2000) FIF [fibroblast growth factor-2 (FGF-2)-interacting-factor], a nuclear putatively antiapoptotic factor, interacts specifically with FGF-2 Mol. Endocrinol. 14,1709-1724[Abstract/Free Full Text]
  4. Morris, E. J., Michaud, W. A., Ji, J-Y., Moon, N-S., Rocco, J. W., Dyson, N. J. (2006) Functional identification of Api5 as a suppressor of E2F-dependent apoptosis in vivo PLoS Genet. 2,e196[CrossRef][Medline]
  5. Bugler, B., Amalric, F., Prats, H. (1991) Alternative initiation of translation determines cytoplasmic or nuclear localization of basic fibroblast growth factor Mol. Cell. Biol. 11,573-577[Abstract/Free Full Text]
  6. Yu, P. J., Ferrari, G., Galloway, A. C., Mignatti, P., Pintucci, G. J. (2007) Basic fibroblast growth factor (FGF-2): the high molecular weight forms come of age J. Cell. Biochem. 100,1100-1108[CrossRef][Medline]
  7. Menzel, T., Rahman, Z., Calleja, E., White, K., Wilson, E. L., Wieder, R., Gabrilove, J. (1996) Elevated intracellular level of basic fibroblast growth factor correlates with stage of chronic lymphocytic leukemia and is associated with resistance to fludarabine Blood 87,1056-1063[Abstract/Free Full Text]
  8. Krejci, P., Dvorakova, D., Krahulcova, E., Pachernik, J., Mayer, J., Hampl, A., Dvorak, P. (2001) FGF-2 abnormalities in B cell chronic lymphocytic and chronic myeloid leukemias Leukemia 15,228-237[CrossRef][Medline]




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