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Blood, 15 March 2002, Vol. 99, No. 6, pp. 2278-2280
CORRESPONDENCE
To the editor:
Immunohistochemical localization of phosphorylated AKT in
multiple myeloma
We read with interest the article by Hsu et al1
analyzing the activation status of AKT in plasma cells from patients
with multiple myeloma (MM) or monoclonal gammopathy of
undetermined significance (MGUS). Recent studies based on MM cell lines
indicate that the phosphatidylinositol 3-kinase (PI3K) signaling
pathway plays a positive role in the survival of myeloma
cells.2 Although various cellular intermediary proteins
are activated by PI3K, recent studies suggest that AKT/PKB activity
alone is sufficient to block apoptosis. Possible activators of the
AKT/PKB signaling pathways in MM include several growth factors such as
insulinlike growth factor-1, epidermal growth factor, basic fibroblast
growth factor, interleukin-3 (IL-3), IL-6, and macrophage
colony-stimulating factor.3 Alternatively, loss of the
tumor suppressor gene, PTEN, may also promote AKT signal activation.
Regardless of the initiating stimulus, full activation of AKT requires
phosphorylation at Thr 308 and Ser 473 by the protein kinases, PDK1
and PDK2. Hsu et al analyzed the phosphorylation status of AKT by using an
antibody that recognizes the phosphorylation site of AKT at Ser473
(pSer473-AKT) and demonstrated primarily a cytoplasmic membrane-specific staining pattern in MM cells. We have also analyzed the expression pattern of pSer473-AKT in 18 MM patients. By
immunohistochemical staining with an anti-pSer473-AKT antibody (Cell
Signaling Technology, Beverly, MA), we found expression of
phosphorylated-AKT in 16 of 18 patients, indicative of constitutively
phosphorylated-AKT in primary MM cells. However, unlike the findings of
Hsu et al, the majority of our samples showed marked nuclear expression
and weaker cytoplasmic reactivity in the plasma cells (Figure
1). Interestingly, plasma cells from 8 patients demonstrated a marked nucleolar staining pattern. Overall, our
findings were consistent with previous reports demonstrating nuclear
localization of phosphorylated-AKT following
activation.4,5 In addition to its well recognized role at
the plasma membrane, AKT is also known to be important in nuclear
transduction.3 Furthermore, AKT has been shown to migrate
to the nucleus following receptor activation as seen with the B-cell
antigen receptor in B lymphocytes.6 Once in the nucleus,
activated AKT is believed to influence the functions of several
regulatory proteins, such as AFX/Forkhead transcription factors,
primarily through regulation of their subcellular
localization.7,8
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| Figure 1.
Immunohistochemical analysis of
pS473-AKT in MM patients.
(A-C) Bone marrow biopsies obtained from MM patients demonstrate
immunopositivity for pSer473-AKT in plasma cells. Biopsies were fixed
in 10% zinc formalin for 2 hours, subsequently decalcified in
Decalcifier II (Surgipath Medical Industries, Richmond, IL) for 2 hours
and processed by a standard automated processor. Four µM sections
mounted on glass slides underwent microwave antigen retrieval process
in citrate buffer, subsequently incubated with anti-pSer473-AKT
antibody at 1/100 dilution overnight at 4°C similar to described
previously.9 Slides were then stained with a
streptavidin/peroxidase detection system.9 Note that
panels A and C illustrate prominent nuclear and nucleolar staining in
the neoplastic plasma cells. Magnification × 500.
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Because IL-6 is known to be
an important cytokine for myeloma cell survival and is known to mediate
phosphorylation of AKT, we analyzed the effect of AKT inhibitors
(wortmannin and LY294 002) on the IL-6-dependent human myeloma cell
line U266. Similar to the analysis of other MM cell lines, both AKT
inhibitors caused marked apoptosis as detected by annexin/7-AAD
staining (Figure 2).2 These
results indicate that IL-6-mediated activation of AKT/PKB signaling is
important for cellular survival of plasma cells in MM.
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| Figure 2.
Effects of AKT inhibitors on U266 cell line.
(A) Control. Exposure to the AKT inhibitors (panel B, wortmannin, 10 µM/L; panel C, LY294002, 10 µM/L) resulted in significantly
increased apoptosis as indicated in each histogram. U266 cells were
incubated with/without the presence of each inhibitor for 72 hours.
Similar to our previous study, apoptosis was quantified by annexin/7AAD
staining, indicating early and late apoptosis,
respectively.9
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In summary, our study confirms that AKT plays a significant role in MM
cell survival. However, the predominant immunohistochemical localization pattern of phosphorylated-AKT differs from that reported by Hsu et al. This is most likely related to the differences in the
techniques used for pSer473-AKT detection, including the source of
antibody, fixation (Bouin vs formalin), and utilization of antigen
retrieval. Overall, our data and those reported by Hsu et al indicate
that overexpression and activation of AKT plays a significant role in
MM cell survival. Thus selective inhibitors that specifically target
the AKT signaling pathway may have important future therapeutic
implications in the treatment of patients with MM.
Serhan Alkan and Keith F. Izban
Correspondence: Serhan Alkan, Department of Pathology, Loyola
University Medical Center, 2160 South First Ave, Maywood, IL 60153;
e-mail: salkan{at}lumc.edu
References
1.
Hsu JH, Shi Y, Krajewski S, et al.
The AKT kinase is activated in multiple myeloma tumor cells.
Blood.
2001;98:2853-2855[Abstract/Free Full Text].
2.
Tu Y, Gardner A, Lichtenstein A.
The phosphatidylinositol 3-kinase/AKT kinase pathway in multiple myeloma plasma cells: roles in cytokine-dependent survival and proliferative responses.
Cancer Res.
2000;60:6763-6770[Abstract/Free Full Text]
3.
Datta SR, Brunet A, Greenberg ME.
Cellular survival: a play in three Akts.
Genes Dev.
1999;13:2905-2927[Free Full Text].
4.
Borgatti P, Martelli AM, Bellacosa A, et al.
Translocation of Akt/PKB to the nucleus of osteoblast-like MC3T3-E1 cells exposed to proliferative growth factors.
FEBS Lett.
2000;477:27-32[CrossRef][Medline]
[Order article via Infotrieve].
5.
Kurose K, Zhou XP, Araki T, Cannistra SA, Maher ER, Eng C.
Frequent loss of PTEN expression is linked to elevated phosphorylated Akt levels, but not associated with p27 and cyclin D1 expression, in primary epithelial ovarian carcinomas.
Am J Pathol.
2001;158:2097-2106[Abstract/Free Full Text].
6.
Astoul E, Watton S, Cantrell D.
The dynamics of protein kinase B regulation during B cell antigen receptor engagement.
J Cell Biol.
1999;145:1511-1520[Abstract/Free Full Text].
7.
Takaishi H, Konishi H, Matsuzaki H, et al.
Regulation of nuclear translocation of Forkhead transcription factor AFX by protein kinase B.
Proc Natl Acad Sci U S A.
1999;96:11836-11841[Abstract/Free Full Text].
8.
Brunet A, Bonni A, Zigmond MJ, et al.
Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor.
Cell.
1999;96:857-868[CrossRef][Medline]
[Order article via Infotrieve].
9.
Ni H, Ergin M, Huang Q, et al.
Analysis of expression of nuclear factor kappaB (NF-kappaB) in multiple myeloma: downregulation of NF-kappaB induces apoptosis.
Br J Haematol.
2001;115:279-286[CrossRef][Medline]
[Order article via Infotrieve]
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