We read with interest the article by Hsu et al1analyzing 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 (Figure1). 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 

Fig. 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.

Fig. 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.

Fig. 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 

Fig. 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.

1
Hsu
JH
Shi
Y
Krajewski
S
et al
The AKT kinase is activated in multiple myeloma tumor cells.
Blood.
98
2001
2853
2855
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.
60
2000
6763
6770
3
Datta
SR
Brunet
A
Greenberg
ME
Cellular survival: a play in three Akts.
Genes Dev.
13
1999
2905
2927
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.
477
2000
27
32
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.
158
2001
2097
2106
6
Astoul
E
Watton
S
Cantrell
D
The dynamics of protein kinase B regulation during B cell antigen receptor engagement.
J Cell Biol.
145
1999
1511
1520
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.
96
1999
11836
11841
8
Brunet
A
Bonni
A
Zigmond
MJ
et al
Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor.
Cell.
96
1999
857
868
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.
115
2001
279
286
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