Stability of Phosphoprotein as a Biological Marker of Tumor Signaling

Materials and Methods

Human tumor xenografts. HT-29 colon cancer cells were obtained from the American Tissue Type Collection (Rockville, MD) and tested to be Mycoplasma-free using a PCR ELISA kit (Roche Diagnostics, Inc., Indianapolis, IN). The cells were grown in humidified 95% air, 5% CO₂ at 37°C in DMEM supplemented with 10% fetal bovine serum. Approximately 10⁷ HT-29 cells were injected s.c. into the flanks of scid mice and the tumors allowed to grow to 250 to 300 mm³. The animals were killed and tumors rapidly excised and either frozen immediately in liquid N₂ or fixed in 5% buffered formalin, in both cases as ∼5 mm³ fragments. The remaining tumor was allowed to stand at room temperature for various times before freezing or fixation in the same way.

Human tumors. In accordance with local Institutional Review Board regulations and after obtaining patient informed consent, small diagnostic biopsies or surgically resected specimens were obtained from patients with unresectable or metastatic adenocarcinoma of the esophagus or gastroesophageal junction as part of a Southwest Oncology Group Trial 0127. The samples were typical tumor bank material obtained by multiple participating institutions collected between September 2002 and May 2004. The biopsies were fixed immediately in 10% buffered formalin. The resected specimens were processed for routine pathologic examination with an indeterminate amount of time before fixation in 10% buffered formalin.

Immunostaining for phospho-Ser⁴⁷³-Akt. Formalin-fixed, paraffin-embedded tissue sections of 4 μm thickness were cut from the blocks, deparaffinized through xylenes and alcohols, blocked in 4% goat serum in PBS for 30 minutes, and treated with a 1:50 dilution of purified anti-phospho-Ser⁴⁷³-Akt rabbit polyclonal antibody (Cell Signaling Technology, Beverly, MA) for 15 hours at 4°C. They were then stained on a Ventana ES automated slide stainer using a Basic DAB Detection kit (Ventana Medical Systems, Tucson, AZ).

Western blotting. The tumor xenografts were homogenized in 50 mmol/L HEPES buffer (pH 7.5), 50 mmol/L NaCl, 1% Nonidet P40, 0.25% sodium deoxycholate, 0.2 mmol/L sodium fluoride, 0.2 mmol/L sodium PPi, and 0.2 mmol/L sodium vanadate. Fifty micrograms of total cell lysate protein were boiled for 5 minutes, loaded on a 12% acrylamide/bisacrylamide gel, and separated by electrophoresis at 160 V for 40 minutes. Proteins were electrophoretically transferred to a polyvinylidene fluoride membranes, preincubated with a blocking buffer of 137 mmol/L NaCl, 2.7 mmol/L KCl, 897 mmol/L CaCl₂, 491 mmol/L MgCl₂, 3.4 mmol/L Na₂HPO₄, 593 mmol/L KH₂PO₄, and 5% bovine serum albumin, and incubated overnight with rabbit purified anti-phospho-Ser⁴⁷³-Akt antibody or anti-Akt antibody (Cell Signaling Technology). Detection used donkey anti-rabbit IgG peroxidase coupled secondary antibody and the Renaissance chemiluminescence system on Kodak X-Omat Blue XB films. Bands were quantified using Eagle Eye software (Stratagene Corp., La Jolla, CA). Tumor Akt activity was expressed as the ratio of phospho-Ser⁴⁷³-Akt to total Akt.

Results

Immunohistochemical staining in human tumor samples. Immunohistochemical staining for phospho-Ser⁴⁷³-Akt in human adenocarcinoma tumor samples gave variable results (Fig. 1A). Evaluable staining was seen only in tumor samples that were obtained as biopsies (9 of 13), whereas no staining was observed in tumors obtained as surgically resected samples (0 of 15). Therefore, we investigated the stability of phospho-Ser⁴⁷³-Akt in HT-29 human colon cancer xenograft samples fixed immediately upon excision from the scid mouse or allowed to stand at room temperature before fixation (Fig. 1B). Whereas phospho-Ser⁴⁷³-Akt staining was apparent in the fresh sample, there was no staining in tissue that had been allowed to stand for 30 to 60 minutes.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 1.

Phospho-AKT in human gastroesophageal tumors and HT-29 colon cancer xenografts measured by immunohistochemical staining. Staining used phospho-Ser⁴⁷³-Akt antibody. A, patient tumor samples. 1 and 2 are two surgically resected specimens and 3 and 4 are two biopsy specimens. B, HT-29 human tumor xenografts were excised from scid mice and kept at room temperature for the times shown. Small pieces were fixed in 5% formalin for immunohistochemistry. Each section also includes in the upper right-hand quadrant an on-slide control of HT-29 colon cancer cells stained for phospho-Ser⁴⁷³-Akt.

Western blotting in tumor xenografts. To distinguish between a loss of phospho-Ser⁴⁷³-Akt and total Akt and to obtain a more accurate time course, we carried out Western blotting studies using HT-29 human tumor xenografts that were allowed to stand at room temperature before rapid freezing in liquid N₂ (Fig. 2). There was a slow decrease in total Akt with a half-life of 180 minutes and a rapid decrease in phospho-Ser⁴⁷³-Akt with a half-life of 20 minutes.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 2.

Stability of phospho-Akt in HT-29 human colon cancer xenografts. HT-29 human tumor xenografts were excised from scid mice and kept at room temperature for the times shown. Small pieces were then rapidly frozen in liquid N₂ for Western blotting. A, phospho-Ser⁴⁷³-Akt and total Akt measured by Western blotting. B, time course of the loss of total Akt (•) and phospho-Ser⁴⁷³-Akt (○) relative to total-Akt.

Collecting human surgical samples. With the information that there was rapid loss of phospho-Ser⁴⁷³-Akt staining, we attempted the rapid collection of a surgically resected human colon cancer specimen. The most rapid collection we could achieve between clamping of the blood supply by the surgeon and release of the tissue by the pathologist was 20 minutes. There was no detectable phospho-Ser⁴⁷³-Akt staining in the samples. The fact that the tumor was at body temperature for a good part of this time may have contributed to the more rapid loss of phospho-Ser⁴⁷³-Akt staining than in the tumor xenograft samples at room temperature.

Discussion

Phosphorylated proteins are frequently used to assess the activity of intracellular signaling pathways and the effects of cancer drugs to inhibit these pathways (2). When this is done in human tumor samples, the speed with which the tumor specimen can be fixed for immunohistochemical staining or Western blotting can have a major effect on the strength of the signal that is seen. We encountered a striking difference in the phospho-Ser⁴⁷³-Akt immunostaining between biopsies where staining was strong and postoperative surgical samples where staining was absent. We found that the half-life of phospho-Ser⁴⁷³-Akt measured by Western blotting in human HT-29 human colon tumor xenografts at room temperature was 20 minutes, whereas total Akt was lost with a half-life of 180 minutes. We had seen a clear correlation between phospo-Ser-Akt staining and whether human adenocarcinoma samples were obtained as biopsies or as surgical specimens, with detection only being seen in biopsies. Biopsies are taken fresh from tumors and likely to be fixed relatively rapidly. Surgical tumor specimens have their blood supply tied and there may be many minutes in the body at 37°C before the tumor is removed and fixed; in our hands, this took at least 20 minutes. Presumed dephosphorylation at 37°C is even faster than at room temperature, although this was not measured directly. The stability of phosphoproteins may depend on the protein being studied and on the tissue or tumor type. However, rapid processing would seem to be essential for any study using phosphoprotein to measure signaling activity or drug inhibition and postoperative surgical samples may be of limited value for measuring phospho-Akt levels.