Identification of phosphoprotein NP33 as a nucleus-associated ribosomal S6 protein and its phosphorylation in hematopoietic cells

Exposure of HL-60 promyelocytes to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate increased incorporation of 32P into a Mr approximately 33,000 protein (NP33) found in the nuclear matrices prepared by treating cells with Triton X-100, nucleases, and 2 M NaCl (D. E. Macfarlane, J. Biol. Chem., 261: 6947-6953, 1986). We now report that 12-O-tetradecanoylphorbol-13-acetate causes phosphorylation of NP33 in U937, K562, HEL, Molt-3, and Raji cell lines, all of which are rapidly proliferating cells of hematopoietic origin. 12-O-Tetradecanoylphorbol-13-acetate caused a lesser degree of NP33 phosphorylation in peripheral blood lymphocytes and monocytes and none in granulocytes or platelets. The incorporation of 32P into NP33 was complete in about 10 min, and it was prevented or reversed by staurosporin, indicating that NP33 is continuously phosphorylated and dephosphorylated. NP33 was purified to homogeneity from Triton X-100-washed nuclei or whole cells by extraction with H2SO4, acetone precipitation, and preparative two-dimensional gel electrophoresis. The amino-terminal amino acid sequence of NP33 appears to be the same as that of ribosomal S6 protein. NP33 appears to be S6 protein copurifying with the nuclear matrix.     

Phosphatidylinositol 3-kinase activity in epidermal growth factor-stimulated matrix metalloproteinase-9 production and cell surface association

Activation of the epidermal growth factor (EGF) receptor regulates many processes associated with metastasis, including modulation of cell:cell and cell:substrate interactions, production of matrix-degrading proteinases, and cellular migration. We have demonstrated previously that EGF stimulates migration and matrix metalloproteinase (MMP)-9-dependent invasion of ovarian cancer cells. In this study, we compare the roles of EGF-induced phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) activities in regulation of cellular responses associated with ovarian tumor cell metastasis. Inhibition of PI3K and MAPK activity impairs EGF-stimulated cell migration, in vitro invasion, and MMP-9 production. PI3K activity is not required for growth factor disruption of cell:cell junctions, whereas inhibitors of extracellular signal-regulated kinase (ERK)1/ERK2 activation and p38 MAPK activity block EGF-dependent junction dissolution. EGF promotes pro-MMP-9 binding to the cell surface through a mechanism that is independent of extracellular enzyme concentration. Interestingly, inhibition of PI3K activity abolishes EGF-induced cell surface association of pro-MMP-9, whereas inhibitors of MAPKs only partially block the response. These data suggest that EGF receptor activation promotes a PI3K-dependent induction of a cell surface pro-MMP-9 binding component that may facilitate gelatinase-mediated cellular invasion and supports an expanded role for elevated PI3K activity in cellular responses associated with ovarian tumor metastasis. In addition, our findings support the hypothesis that divergent kinase activities regulate distinct cellular events associated with growth factor-induced invasion of ovarian cancer cells.     

Increase of epidermal growth factor-stimulated cell-cycle progression and induction of thermotolerance by heat shock: temperature and time relationship

When quiescent confluent cultures were incubated at increased temperature and then incubated at 37 degrees C prior to a second increase of temperature (46 degrees C) it appeared that heat-induced morphological alteration and ability to proliferate could be influenced by the previous thermal history of the cells. Incubations for 20 min in a temperature range of 41-46 degrees C caused cells to develop thermo-tolerance within 3 h of incubation at 37 degrees C. Confluent quiescent Swiss mouse 3T3 cells were incubated at 41.8, 43.7 or 45.6 degrees C and then reincubated at 37 degrees C to determine the effects of heat shock on the mitogenic effects of epidermal growth factor (EGF). Preincubation at 43.7 degrees C or 45.6 degrees C enhanced stimulation of G1-S progression by EGF. Preincubation at 43.7 degrees C markedly increased the rate at which cells enter the S phase without changing the length of the lag phase. A comparison of the duration of incubation at 43.7 degrees C for potentiation of EGF-induced DNA synthesis and that for induction of thermotolerance showed that a similar time interval for induction of effect could be implied.     

Increase of epidermal growth factor-stimulated cell-cycle progression and induction of thermotolerance by heat shock: Temperature and time relationship

When quiescent confluent cultures were incubated at increased temperature and then incubated at 37°C prior to a second increase of temperature (46°C) it appeared that heat-induced morphological alteration and ability to proliferate could be influenced by the previous thermal history of the cells. Incubations for 20 min in a temperature range of 41–46°C caused cells to develop thermotolerance within 3 h of incubation at 37°C.

Confluent quiescent Swiss mouse 3T3 cells were incubated at 41.8, 43.7 or 45.6°C and then reincubated at 37°C to determine the effects of heat shock on the mitogenic effects of epidermal growth factor (EGF). Preincubation at 43.7°C or 45.6°C enhanced stimulation of G₁-S progression by EGF. Preincubation at 43.7°C markedly increased the rate at which cells enter the S phase without changing the length of the lag phase. A comparison of the duration of incubation at 43.7°C for potentiation of EGF-induced DNA synthesis and that for induction of thermotolerance showed that a similar time interval for induction of effect could be implied.     

Bioassay using epidermal keratinocytes to determine phosphorylation status of the epidermal growth factor receptor in distant neoplasms

We developed a bioassay to evaluate the phosphorylation status of a fibrosarcoma following systemic administration of the protein tyrosine kinase inhibitor PKI 166. Samples of subcutaneous fibrosarcomas and distant skin were fixed in formalin, sectioned, and stained with several fluorescent antibodies against the epidermal growth factor receptor (EGF-R) and phosphorylated EGF-R. In mice given different doses of PKI 166, the dose-dependent inhibition of phosphorylation of EGF-R in epidermal keratinocytes paralleled that in fibrosarcomas growing subcutaneously, suggesting that skin biopsies can be used as surrogate tissues for distant neoplasms to determine the phosphorylation status of protein tyrosine kinase receptors.     

Relationship between 43 kDa epidermal growth factor-related clonogenic activity and clinical parameters for breast cancer.

The clonogenic activity of a previously purified 43 kDa EGF-related protein (16) was estimated in the urine of breast cancer patients. Increase of activity was statistically significant in lymph node-positive patients, in a group of patients with larger carcinomas, with accelerated tumor growth, in premenopausal patients and younger age and in estrogen receptor negative patients. In 31 patients the activity was estimated during polychemotherapy before surgery. Differences between the values at the begin and the end of treatment were compared between all groups by the Kruskal-Wallis test (p = 0.02). Patients with progressive disease showed increasing activities (mean values from 315 to 811) while in those with complete remission activity decreased (from 449 to 213). Partial- and non responders showed no change. In a long-term follow-up study with 25 patients the pre- and postoperative activities were estimated. In 17 of 18 patients who had no local recurrence or metastasis the activity declined after surgery, whereas in 6 of 7 patients who died, activity increased 2-5 fold prior to death. A life table analysis with a total of 101 patients revealed a trend to shorter survival in the group with higher activity (p = 0.042). These observations suggest a role for the EGF-like growth factor activity in the expression of the malignant phenotype and may have significance for breast cancer diagnosis and prognosis.     

Mechanism of antimitogenic action of vitamin D in human colon carcinoma cells: relevance for suppression of epidermal growth factor-stimulated cell growth.

Because the efficacy of 1alpha,25-dihydroxyvitamin D3 [1alpha,25-(OH)2D3] in treatment of colon cancer might critically depend on its ability to specifically counteract epidermal growth factor (EGF)-stimulated tumor cell growth, we utilized human colon adenocarcinoma-derived cells in primary culture as well as the Caco-2 cell line to elucidate possible sites of interaction of 1alpha,25-(OH)2D3 with signaling from EGF receptor activation. In both types of colon cancer cells investigated, 10(-8) M 1alpha,25-(OH)2D3 reduced basal cell proliferation by about 50%, and prevented any rise in proliferation when colon cancer cells were treated with 25 ng/ml EGF: this can be explained by a marked inhibitory effect of 1alpha,25-(OH)2D3 on EGFR mRNA and protein expression. The steroid hormone also seemingly promotes EGF-induced internalization of apical and basolateral membrane EGFR. In addition, 1alpha,25-(OH)2D3 significantly reduced basal and EGF-stimulated expression of cyclin D1 at the mRNA and protein level in primary cultures as well as in the Caco-2 cell line. The ability of 1alpha,25-(OH)2D3 to interfere with a key event in cell cycle control and thereby to block mitogenic signaling from EGF could be seen as advantageous for the potential use of vitamin D compounds in colon cancer therapy.     

Demonstration of a 65 kDa tumor-specific phosphoprotein in urine and serum of rats with N-methyl-N-nitrosourea-induced mammary adenocarcinomas

Polyclonal antibodies against a 65 kDa tumor-associated phosphoprotein(p⁶⁵) were used to develop an ELISA to analyze the presenceof p⁶⁵ in urine and serum of rats bearing N-methyl-N-nitrosourea-inducedmammary gland adeno-carcinomas. Highly purified rat p⁶⁵ wasadded to normal urine and serum to establish a quantitativestandard curve with the average correlation coefficient being0.98 and 0.99 respectively. All samples of urine and serum obtainedfrom different carcinoma-bearing rats showed p65 concentrationsabove the normal levels found in the control urine and sera.The correlation coefficient between tumor burden and p⁶⁵ concentrationin urine and serum was 0.65 and 0.77 respectively. The averagelevels of p⁶⁵ in normal urine and normal serum were 37.0 ±32.0 and 48.0 ± 38.0 ng/ml respectively. In the caseof urine obtained from rats bearing mammary adenocarcinomas,the mean p⁶⁵ level was 119.0 ± 35.9 ng/ml and their serumlevel was 225.4 ± 67.5 ng/ml. Sensitivity, specificityand predictive value for serum and urine marker elevation were78.5, 70.0 and 78.5% respectively. Following in vitro phosphorylationof concentrated urinary proteins, isoelectrofocusing, SDS-PAGEand autoradiography, a phosphorylated form of the 65 kDa proteinwith a pI of 5.8 was identified in the urine of tumor-bearingrats. This phosphoprotein bound to an antiphosphotyrosine monoclonalantibody and an anti-p⁶⁵ polyclonal as determined by Westernblot analysis. Using the anti-p⁶⁵ antibodies in an immunoprecipitationprocedure, the main radio- and immunoactive band of 65 kDa andtwo lower mol. wt bands of 50 and 41 kDa, apparently representingdegradation products of p⁶⁵, were identified after in vitroand in vivo phosphorylation of urinary proteins obtained frommammary carcinoma-bearing rats.     

Inhibition of ligand-induced activation of epidermal growth factor receptor tyrosine phosphorylation by curcumin

We explored the regulation of epidermal growth factor (EGF)-mediatedactivation of EGF receptor (EGF-R) phosphorylation by curcumin(diferuloyl-methane), a recently identified kinase inhibitor,in cultured NIH 3T3 cells expressing human EGF-R. Treatmentof cells with a saturating concentration of EGF for 5–15min induced increased EGF-R tyrosine phosphorylation by 4- to11-fold and this was inhibited in a dose- and time-dependentmanner by up to 90% by curcumin, which also inhibited the growthof EGF-stimulated cells. There was no effect of curcumin treatmenton the amount of surface expression of labeled EGF-R and inhibitionof EGF-mediated tyrosine phosphorylation of EGF-R by curcuminwas mediated by a reversible mechanism. In addition, curcuminalso inhibited EGF-induced, but not bradykinin-induced, calciumrelease. These findings demonstrate that curcumin is a potentinhibitor of a growth stimulatory pathway, the ligand-inducedactivation of EGF-R, and may potentially be useful in developinganti-proliferative strategies to control tumor cell growth.     

Sulindac sulfide inhibits epidermal growth factor-induced phosphorylation of extracellular-regulated kinase 1/2 and Bad in human colon cancer cells

Colorectal cancer is the second leading cause of cancer death in the United States. Nonsteroidal anti-inflammatory drugs including sulindac are promising chemopreventive agents for colorectal cancer. Sulindac and selective cyclooxygenase (COX)-2 inhibitors cause regression of colonic polyps in familial polyposis patients. Sulindac induces apoptotic cell death in cancer cells in vitro and in vivo. In tumor cells, activation of extracellular-regulated kinase (ERK) 1/2 results in phosphorylation of several ERK1/2 effectors, including the proapoptotic protein Bad. Phosphorylation of Ser112 by ERK1/2 inactivates Bad and protects the tumor cell from apoptosis. Sulindac metabolites and other nonsteroidal anti-inflammatory drugs selectively inhibit ERK1/2 phosphorylation in human colon cancer cells. In this study we show that epidermal growth factor (EGF) strongly induces phosphorylation of ERK1/2 and Bad in HT29 colon cancer cells. EGF-stimulated phosphorylation of ERK and Bad is blocked by pretreatment with U0126, a selective MAP kinase kinase (MKK)1/2 inhibitor. Similarly, pretreatment with sulindac sulfide blocks the ability of EGF to induce ERK1/2 and Bad phosphorylation, but also down-regulates total Bad but not ERK1/2 protein levels. The ability of sulindac to block ERK1/2 signaling by the EGF receptor may account for at least part of its potent growth-inhibitory effects against cancer cells.     

Inhibition of focal adhesion kinase expression or activity disrupts epidermal growth factor-stimulated signaling promoting the migration of invasive human carcinoma cells

Elevated focal adhesion kinase (FAK) expression in human tumor cells has been correlated with an increased cell invasion potential. In cell culture, studies with FAK-null fibroblasts have shown that FAK function is required for cell migration. To determine the role of elevated FAK expression in facilitating epidermal growth factor (EGF)-stimulated human adenocarcinoma (A549) cell motility, antisense oligonucleotides were used to reduce FAK protein expression >75%. Treatment of A549 cells with FAK antisense (ISIS 15421) but not a mismatched control (ISIS 17636) oligonucleotide resulted in reduced EGF-stimulated p130(Cas)-Src complex formation, c-Jun NH(2)-terminal kinase (JNK) activation, directed cell motility, and serum-stimulated cell invasion through Matrigel. Because residual FAK protein in ISIS 15421-treated A549 cells was highly phosphorylated at the Tyr-397/Src homology (SH)2 binding site, expression of the FAK COOH-terminal domain (FRNK) was also used as an inhibitor of FAK function. Adenoviral-mediated infection and expression of FRNK promoted FAK dephosphorylation at Tyr-397, resulted in reduced EGF-stimulated JNK as well as extracellular-regulated kinase 2 (ERK2) kinase activation, inhibited matrix metalloproteinase-9 (MMP-9) secretion, and potently blocked both random and EGF-stimulated A549 cell motility. Equivalent expression of a FRNK (S-1034) point-mutant that did not promote FAK dephosphorylation also did not affect EGF-stimulated signaling or cell motility. Dose-dependent reduction in EGF-stimulated A549 motility was observed with the PD98059 MEK1 inhibitor and the batimastat (BB-94) inhibitor of MMP activity, but not with the SB203580 inhibitor of p38 kinase. Finally, comparisons between normal, FAK-null, and FAK-reconstituted fibroblasts revealed that FAK enhanced EGF-stimulated JNK and ERK2 kinase activation that was required for cell motility. These data indicate that FAK functions as an important signaling platform to coordinate EGF-stimulated cell migration in human tumor cells and support a role for inhibitors of FAK expression or activity in the control of neoplastic cell invasion.     

The radioprotector O-phospho-tyrosine stimulates DNA-repair via epidermal growth factor receptor- and DNA-dependent kinase phosphorylation.

BACKGROUND AND PURPOSE: Purpose of the study was to elucidate the underlying molecular mechanism of the radioprotector O-phospho-tyrosine (P-Tyr). METHODS: Molecular effects of P-Tyr at the level of EGFR responses were investigated in vitro with bronchial carcinoma cell line A549. Nuclear EGFR transport and DNA-PK activation were quantified after Western blotting. Residual DNA-damages were quantified by help of gammaH(2)AX focus assay. RESULTS: As determined by dose-response curves, treatment of cells with P-Tyr for 16h before irradiation results in radioprotection. Simultaneous treatment with EGFR blocking antibody Cetuximab abolished P-Tyr associated radioprotection. At the molecular level P-Tyr mediated a general phosphorylation of EGFR and a pronounced phosphorylation of nuclear EGFR at residue Thr No. 654, also observed after treatment with ionizing radiation. This phosphorylation was associated with nuclear EGFR accumulation. Moreover, P-Tyr-triggered EGFR nuclear accumulation was associated with phosphorylation of DNA-PK at Thr 2609. This activated form of DNA-PK was not DNA associated, but after radiation, DNA binding increased, particularly after P-Tyr pre-treatment. These molecular effects of P-Tyr resulted in a reduction of residual DNA-damage after irradiation. CONCLUSIONS: Radioprotection by P-Tyr is mediated through its stimulation of nuclear EGFR transport and concurrent, but DNA-damage independent, activation of DNA-PK. Thus, subsequent irradiation results in increased binding of DNA-PK to DNA, improved DNA-repair and increased cell survival.     

ERK phosphorylation predicts synergism between gemcitabine and the epidermal growth factor receptor inhibitor AG1478

Background

Clinical trials combining epidermal growth factor receptor (EGFR) inhibitors with gemcitabine-based chemotherapy in non-small cell lung cancer (NSCLC) have not produced a survival advantage. This may be caused by antagonism between the two drugs or mutations that promote such, possibly RAS mutation. Furthermore, ERK, a critical growth regulator downstream of RAS, may play a role. This study aimed to explore the relationship between ERK, synergy/antagonism and cell cycle arrest in combination treatment.

Methods

A549 (mutant KRAS), H322 (wildtype KRAS) and siRNA-mediated KRAS knockdown A549 were treated with gemcitabine and/or the EGFR inhibitor AG1478 and analyzed with median effect analysis. Cell cycle distribution and ERK phosphorylation were assessed using flow cytometry and ELISA, respectively. Effect on cytotoxicity after ERK inhibition by U0126 was also assessed.

Results

Cytotoxic interaction was dose dependent with antagonism at high dose AG1478. G1 arrest was observed with both high dose AG1478 and high dose gemcitabine and therefore was inconsistently associated with antagonism. Furthermore, ERK phosphorylation was increased by gemcitabine and its suppression by AG1478 was related to antagonism particularly in H322. ERK's effect in antagonism was further confirmed by using U0126. Greater antagonism was observed in the KRAS mutant cell line and KRAS knockdown by siRNA resulted in increased sensitivity to AG1478 as well as combination treatment.

Conclusion

Our findings are consistent with a model in which ERK phosphorylation favors synergy and the outcome depends on the balance between gemcitabine-induced and AG1478-inhibited ERK phosphorylation. KRAS mutation confers resistance to AG1478 as well as combination treatment.     

SHP-2-dependent mitogen-activated protein kinase activation regulates EGFRvIII but not wild-type epidermal growth factor receptor phosphorylation and glioblastoma cell survival

In human glioblastomas, the most common mutation of epidermal growth factor receptor (EGFR) is an in-frame deletion of an 801-bp sequence in the extracellular domain of EGFR termed EGFRvIII. The EGFRvIII does not bind ligand EGF but has constitutive tyrosine phosphorylation (pTyr) content and kinase activity that result in enhanced transformation, reduced apoptosis, and resistance to therapy. Here we report that the protein tyrosine phosphatase SHP-2 modulates a mitogen-activated protein kinase (MAPK) kinase (MEK)-mediated signaling pathway that regulates EGFRvIII pTyr and cell survival in U87MG.EGFRvIII cells. Overexpression of the phosphatase-inactive form of SHP-2 inhibited EGFRvIII pTyr by decreasing MAPK phosphorylation. Consistent with this, we observed that the MEK inhibitor PD98059, but not the phosphatidylinositol 3'-kinase inhibitor LY294002, inhibited EGFRvIII pTyr. Furthermore, constitutive EGFRvIII pTyr content observed in U87MG, LN229, and U373MG glioblastoma cells, but not in NR6.EGFRvIII fibroblasts, correlated with elevated MAPK levels in these cells. Interestingly, LY294002, but not PD98059, inhibited wild-type EGFR pTyr in response to EGF treatment in U87MG parental cells and in wild-type EGFR-overexpressing U87MG cells. Inhibition of EGFRvIII pTyr by PD98059 was not observed to be phosphorylation site specific. However, LY294002 more specifically inhibited wild-type EGFR pTyr at residues Tyr(992) and Tyr(1068) in the COOH terminus. Treatment of U87MG.EGFRvIII cells with PD98059, but not LY294002, also resulted in increased cell death in response to cisplatin. Collectively, a distinct MEK-mediated pathway in human glioblastoma cells appears to differentially modulate EGFRvIII and wild-type EGFR pTyr, and inhibition of the MAPK pathway sensitizes EGFRvIII-containing human glioblastoma cells to cisplatin-induced cell death.     

The biology of human epidermal growth factor receptor 2

Our understanding of the normal signaling mechanisms and functions of human epidermal growth factor receptor 2 (HER2) and other members of the HER family, namely epidermal growth factor receptor, HER3, and HER4, is growing rapidly. Activation of these receptors results in a diverse array of signals through the formation of homodimeric and heterodimeric receptor complexes; HER2 is the preferred dimerization partner for the other HERs. These oligomeric receptor complexes activate distinct signaling pathways, such as the Ras-MAPK and PI3-kinase pathways. These, in turn, affect various cellular processes. Recent gene deletion experiments in mice point to an important role for HER2 in cardiac and neural development, and evidence from other studies indicates that HER2 is involved in normal breast growth and development. Thus, HER2 is a key component of a complex signaling network that plays a critical role in the regulation of tissue development, growth, and differentiation.