Activated K-RAS increases polyamine uptake in human colon cancer cells through modulation of caveolar endocytosis

Abstract

Endocytic pathways have been implicated in polyamine transport in mammalian cells, but specific mechanisms have not been described. We have shown that expression of a dominant negative (DN) form of the GTPase Dynamin, but not Eps15, diminished polyamine uptake in colon cancer cells indicating a caveolar and nonclathrin uptake mode. Polyamines co-sediment with lipid raft/caveolin-1 rich fractions, of the plasma membrane in a sucrose density gradient. Knock down of caveolin-1 significantly increased polyamine uptake. Conversely, ectopic expression of this protein resulted in diminished polyamine uptake. We also found that presence of an activated K-RAS oncogene significantly increased polyamine uptake by colon cancer cells. This effect is through an increase in caveolin-1 phosphorylation at tyrosine residue 14. Caveolin-1 is a negative regulator of caveolar endocytosis and phosphorylation in a K-RAS dependent manner leads to an increase in caveolar endocytosis. In cells expressing wild type K-RAS, addition of exogenous uPA was sufficient to stimulate caveolar endocytosis of polyamines. This effect was abrogated by the addition of a SRC kinase inhibitor. These data indicate that polyamine transport follows a dynamin-dependent and clathrin-independent endocytic uptake route, and this route is positively regulated by the oncogenic expression of K-RAS in a caveolin-1 dependent manner.

Copyright 2008 Wiley-Liss, Inc.

Figures

Figure 1

Incubation at a low temperature (4°C) inhibits uptake of transferrin, cholera toxin and spermidine in HCT116 cells. (A) HCT116 were plated for 48 h and then incubated at 4° or 37° for 30 min. Transferrin uptake with AlexaFluor 488-conjugated transferrin was assessed, as described in Materials and Methods Section. (B) HCT116 were plated for 48 h and then incubated at 4° or 37° for 30 min. Cholera toxin uptake with AlexaFluor 488-conjugated cholera toxin subunit-B was assessed, as described in Materials and Methods Section. (C) HCT116 were plated for 48 h and then incubated at 4° or 37° for 30 min. Polyamine uptake with radiolabeled spermidine was assessed, as described in Materials and Methods Section.

Figure 2

Treatment with Brefeldin A, an inhibitor of endocytosis, abrogates transferrin, cholera toxin and spermidine uptake in HCT116 cells. (A) HCT116 were plated for 48 h and then incubated with 10 μg/mL Brefeldin A at 37° for 30 min. Transferrin uptake with AlexaFluor 488-conjugated transferrin was assessed, as described in Materials and Methods Section. (B) HCT116 were plated for 48 h and then incubated with 10 μg/mL Brefeldin A at 37° for 30 min. Cholera toxin uptake with AlexaFluor 488-conjugated cholera toxin subunit-B was assessed, as described in Materials and Methods Section. (C) HCT116 were plated for 48 h and then incubated with 10 μg/mL Brefeldin A at 37° for 30 min. Polyamine uptake with radiolabeled spermidine was assessed, as described in Materials and Methods Section.

Figure 3

The macropinocytosis inhibitor, Amiloride, has no effect on transferrin, cholera toxin and spermidine uptake in HCT116 cells. (A) HCT116 were plated for 48 h and then incubated with 10 μM Amiloride at 37° for 30 min. Transferrin uptake with AlexaFluor 488-conjugated transferrin was assessed, as described in Materials and Methods Section. (B) HCT116 were plated for 48 h and then incubated with 10 μM Amiloride at 37° for 30 min. Cholera toxin uptake with AlexaFluor 488-conjugated cholera toxin subunit-B was assessed, as described in Materials and Methods Section. (C) HCT116 were plated for 48 h and then incubated with 10 μM Amiloride A at 37° for 30 min. Polyamine uptake with radiolabeled spermidine was assessed, as described in Materials and Methods Section.

Figure 4

Perturbation of dynamin-dependent endocytosis blocks polyamine uptake in HCT116 cells. (A) HCT116 cells were transfected with DynWT and DynK44A, as described in Materials and Methods Section. Eighteen hours after transfection, cells were serum-starved for 1 h and treated with 10 μL of Alexa-Fluor 633 transferrin for 30 min. Cells were trypsinized and collected for FACS analysis. MFI was calculated after cells were sorted in a BDFacs Aria machine. In all figures, *P-value of <0.01. (B) HCT116 cells were transfected with DynWT and DynK44A. Twenty-four hours after transfection, cells were washed twice with serum free medium. Putrescine uptake was assayed as described in Materials and Methods Section. (C) HCT116 cells were transfected with DynWT and DynK44A. Twenty-four hours after transfection, cells were washed twice with serum free medium. Spermidine uptake was assayed as described in Materials and Methods Section. In all figures, *P-value of 0.01.

Figure 5

Clathrin-dependent endocytosis does not affect polyamine uptake in HCT116 cells. (A) HCT116 cells were transfected with Eps15 WT and Eps15 DN, as described in Materials and Methods Section. Eighteen hours after transfection, cells were serum-starved for 1 h and treated with 10 μL of Alexa-Fluor 633 transferrin for 30 min. Cells were trypsinized and collected for FACS analysis. MFI was calculated after cells were sorted in a BDFacs Aria machine. In all figures, *P-value of <0.01. (B) HCT116 cells were transfected with Eps15 WT and Eps15 DN. Twenty-four hours after transfection, cells were washed twice with serum free medium. Putrescine uptake was assayed as described in Materials and Methods Section. (C) HCT116 cells were transfected with Eps15 WT and Eps15 DN. Twenty-four hours after transfection, cells were washed twice with serum free medium. Spermidine uptake was assayed as described in Materials and Methods Section. In all figures, *P-value of <0.01.

Figure 6

Polyamines associate with lipid raft/caveolin-1 rich fractions in a sucrose density gradient, and their uptake is inhibited with drugs that perturb caveolar endocytosis. (A) HCT116 cells were grown to subconfluency in 150 mm plates. Plates were washed twice with serum free medium and labeled with tritiated spermidine for 15 min at 4°C. Cells were then lyzed, fractions collected and counted as described in Materials and Methods Section. Same experiments were run without the radiolabel. Fractions were collected and resolved on an SDS–PAGE gel and immunoblotted for caveolin-1 and flotillin-1. (B) HCT116 caveolin-1 antisense transfected cells were analyzed as in Figure 6A. (C) HCT116 cells were plated for 48 h. They were then serum starved and treated with various inhibitors of endocytosis for 30–60 min at 37°C. Transferrin uptake with AlexaFluor 488-conjugated transferrin was assessed, as described in Materials and Methods Section. (D) HCT116 cells were plated for 48 h. They were then serum starved and treated with various inhibitors of endocytosis for 30–60 min at 37°C. Cholera toxin uptake with AlexaFluor 488-conjugated cholera toxin subunit-B was assessed, as described in Materials and Methods Section. (E) HCT116 cells were plated for 48 h. They were then serum starved and treated with various inhibitors of endocytosis for 30–60 min at 37°C. Polyamine uptake with radiolabeled putrescine was assessed, as described in Materials and Methods Section. (F) HCT116 cells were plated for 48 h. They were then serum starved and treated with various inhibitors of endocytosis for 30–60 min at 37°C. Polyamine uptake with radiolabeled spermidine was assessed, as described in Materials and Methods Section. In all figures, *, **, and ***P-value of <0.01. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 6

Polyamines associate with lipid raft/caveolin-1 rich fractions in a sucrose density gradient, and their uptake is inhibited with drugs that perturb caveolar endocytosis. (A) HCT116 cells were grown to subconfluency in 150 mm plates. Plates were washed twice with serum free medium and labeled with tritiated spermidine for 15 min at 4°C. Cells were then lyzed, fractions collected and counted as described in Materials and Methods Section. Same experiments were run without the radiolabel. Fractions were collected and resolved on an SDS–PAGE gel and immunoblotted for caveolin-1 and flotillin-1. (B) HCT116 caveolin-1 antisense transfected cells were analyzed as in Figure 6A. (C) HCT116 cells were plated for 48 h. They were then serum starved and treated with various inhibitors of endocytosis for 30–60 min at 37°C. Transferrin uptake with AlexaFluor 488-conjugated transferrin was assessed, as described in Materials and Methods Section. (D) HCT116 cells were plated for 48 h. They were then serum starved and treated with various inhibitors of endocytosis for 30–60 min at 37°C. Cholera toxin uptake with AlexaFluor 488-conjugated cholera toxin subunit-B was assessed, as described in Materials and Methods Section. (E) HCT116 cells were plated for 48 h. They were then serum starved and treated with various inhibitors of endocytosis for 30–60 min at 37°C. Polyamine uptake with radiolabeled putrescine was assessed, as described in Materials and Methods Section. (F) HCT116 cells were plated for 48 h. They were then serum starved and treated with various inhibitors of endocytosis for 30–60 min at 37°C. Polyamine uptake with radiolabeled spermidine was assessed, as described in Materials and Methods Section. In all figures, *, **, and ***P-value of <0.01. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 7

Loss of caveolin-1 increases polyamine uptake in HCT116 cells. (A) HCT116-Mock and Caveolin-1 Antisense transfected cells were grown to sub-confluency and lysed in RIPA buffer. Equal amounts of protein were resolved on SDS–PAGE gels and probed for caveolin-1. (B) HCT116-Mock and Caveolin-1 Antisense transfected cells were plated for 48 h. They were then serum starved for 60 min at 37°C. Cholera toxin uptake with AlexaFluor 488-conjugated cholera toxin subunit-B was assessed, as described in Materials and Methods Section. (C) HCT116-Mock and Caveolin-1 Antisense transfected cells were plated for 48 h. Polyamine uptake with radiolabeled putrescine was assessed, as described in Materials and Methods Section. (D) HCT116-Mock and Caveolin-1 Antisense transfected cells were plated for 48 h. Polyamine uptake with radiolabeled spermidine was assessed, as described in Materials and Methods Section.

Figure 8

Ectopic expression of caveolin-1 decreases polyamine uptake in HT29 cells. (A) HT29-Mock and Caveolin-1 transfected cells were plated for 24 h. Following incubation, IPTG (1 mM) was added for an additional 24 h. Cells were then lysed, and equal amounts of protein were resolved on SDS–PAGE gels and probed for caveolin-1. (B) HT29-Mock and Caveolin-1 transfected cells were plated for 24 h. Following incubation, IPTG (1 mM) was added for an additional 24 h. Polyamine uptake with radiolabeled putrescine was assessed, as described in Materials and Methods Section. (C) HT29-Mock and Caveolin-1 transfected cells were plated for 24 h. Following incubation, IPTG (1 mM) was added for an additional 24 h. Polyamine uptake with radiolabeled spermidine was assessed, as described in Materials and Methods Section.

Figure 9

Activated K-RAS increases polyamine uptake in HCT116 cells, by increasing caveolin-1 tyrosine residue 14 phosphorylation. (A) HCT116 and Hkh2 cells were plated for 48 h. Polyamine uptake with radiolabeled putrescine was assessed, as described in Materials and Methods Section. (B) HCT116 and Hkh2 cells were plated for 48 h. Polyamine uptake with radiolabeled spermidine was assessed, as described in Materials and Methods Section. (C) Caco-Neo and Caco-K-Ras#26 cells were plated for 48 h. Polyamine uptake with radiolabeled putrescine was assessed, as described in Materials and Methods Section.

Figure 10

Activated K-RAS increases caveolin-1 phosphorylation in HCT116 cells in a SRC-dependent manner. (A) HCT116 and Hkh2 cells were plated for 48 h RAS activity status using the Ras Activation Assay Kit (Catalog # 17-218) from Upstate, Millipore Corporation using the manufacturer's protocol, as described in Materials and Methods Section. (B) HCT116 and Hkh2 cells were plated for 48 h. They were then serum starved for 60 min at 37°C. Cholera toxin uptake with AlexaFluor 488-conjugated cholera toxin subunit-B was assessed, as described in Materials and Methods Section. (C,D) HCT116 and Hkh2 cells were grown to sub-confluency and lysed in RIPA buffer. Equal amounts of protein were resolved on SDS–PAGE gels and probed for phospho-caveolin-1, caveolin-1, phospho-Src, and total Src. For PP2 treatment, cells were serum-starved for 1 h. PP2 (5 μM) was added for an additional 30 min before lysis.

Figure 11

Exogenous uPA increases caveolin-1 phosphorylation and polyamine uptake in Hkh2 cells, in a SRC dependent manner. (A) Hkh2 cells were grown to sub-confluency and treated with 50 ng/mL uPA for 30 min. They were lysed in RIPA buffer. Equal amounts of protein were resolved on SDS–PAGE gels and probed for phospho-caveolin-1, caveolin-1, phospho-Src, and total Src. For PP2 treatment, cells were serum-starved for 1 h. PP2 (5 μM) was added for an additional 30 min before lysis. This was followed by uPA treatment for 30 min after which cells were lysed in RIPA buffer. (B) Hkh2 cells were grown to sub-confluency and treated with 50 ng/mL uPA for 30 min. Cholera toxin uptake was carried out as described in Materials and Methods Section. For PP2 treatment, cells were serum-starved for 1 h. PP2 (5 μM) was added for an additional 30 min uptake. (C) Hkh2 cells were plated for 48 h. They were then serum-starved for 1 h after which PP2 (5 μM) was added for 30 min. This was followed by the addition of 50 ng/mL uPA for an additional 30 min. Polyamine uptake with radiolabeled spermidine was assessed, as described in Materials and Methods Section.

Figure 12

Model for an activated K-RAS dependent polyamine uptake in colon cancer cells. 1,2: An activated K-RAS oncogene increases uPAR gene expression. 3: Increased uPAR expression leads to more uPA/uPAR interaction and subsequent SRC activation. 4,5: This in turn increases phosphorylation of caveolin-1 at tyrosine residue 14 and more polyamine internalization via caveolar endocytosis.