22010 Background: De novo and acquired resistance of HER2+ breast cancer (HBC) totrastuzumab (T) is an important clinical problem, yet the molecular basis underlying resistance is not well understood. Mutations in the T binding domain of HER2 and a splicing variant of HER2 lacking exon 16 (del16) have been identified in small studies and postulated to play a role in T resistance. To identify additional mutations and splice variants in HER2, we utilized a sensitive mismatch detection assay to systematically screen the entire HER2 cDNA for such alterations. Methods: Total RNA was isolated from cell lines and frozen primary breast cancer samples and cDNA synthesized using oligo-(dT)20 primers. Seven sets of HER2-specific primers were used to PCR amplify the entire coding region of HER2 cDNA. Using Transgenomic WAVE HPLC, deletions were resolved by size, and point mutations were identified by Surveyor endonuclease. Fractions from peaks of interest were collected for subsequent Sanger sequencing. Results: 18 breast cancer cell lines, 11 primary HBC samples, and 8 normal breast controls were analyzed. No point mutations were identified. However, in addition to the previously described del16 transcript, several novel splice variants were observed in cell lines and primary HBC, including transcripts with deletion of exons 4–7 of the extracellular domain, and transcripts with deletion of exons 20–23 (del20–23) encoding the kinase domain. All of these variants were in-frame. To determine the relevance of these splice variants, we generated T-resistant clones of the cell lines BT-474, MDA-MB453, SKBR3, and ZR75–1. In all T-resistant cell lines except the SKBR3, the level of the del20–23 transcript was markedly lower than that of the parental cell line. Conclusions: In contrast to a previous report, we did not detect mutations in HER2 in a limited survey of HBC samples using a highly sensitive assay. However, several novel splice variants were identified. The expression of del20–23, lacking the kinase domain, was downregulated in T-resistant cell lines, suggesting that this variant may modulate HER2 signaling and T resistance. Functional studies of the protein encoded by this variant are warranted. No significant financial relationships to disclose.
The Epstein-Barr virus BRLF1 and BZLF1 genes are the first viral genes transcribed upon induction of the viral lytic cycle. The protein products of both genes (referred to here as Rta and Zta, respectively) activate expression of other viral genes, thereby initiating the lytic cascade. Among the viral antigens expressed upon induction of the lytic cycle, however, Zta is unique in its ability to disrupt viral latency; expression of the BZLF1 gene is both necessary and sufficient for triggering the viral lytic cascade. We have previously shown that Zta can activate its own promoter (Zp), through binding to two Zta recognition sequences (ZIIIA and ZIIIB). Here we describe mutant Zta proteins that do not bind DNA (referred to as Zta DNA-binding mutants [Zdbm]) but retain the ability to transactivate Zp. Consistent with the inability of these mutants to bind DNA, transactivation of Zp by Zdbm is not dependent on the Zta recognition sequences. Instead, transactivation by Zdbm is dependent upon promoter elements that bind cellular factors. An examination of other viral and cellular promoters identified promoters that are weakly responsive or unresponsive to Zdbm. An analysis of a panel of artificial promoters containing one copy of various promoter elements demonstrated a specificity for Zdbm activation that is distinct from that of Zta. These results suggest that non-DNA-binding forms of some transactivators retain the ability to transactivate specific target promoters without direct binding to DNA.
This is a phase II, multicenter, open-label study of chemotherapy-naïve patients with non-small-cell lung cancer (NSCLC) and age > or = 70 years who were treated with erlotinib and evaluated to determine the median, 1-year, and 2-year survival. The secondary end points include radiographic response rate, time to progression (TTP), toxicity, and symptom improvement.Eligible patients with NSCLC were treated with erlotinib 150 mg/d until disease progression or significant toxicity. Tumor response was assessed every 8 weeks by computed tomography scan using Response Evaluation Criteria in Solid Tumors. Tumor samples were analyzed for the presence of somatic mutations in EGFR and KRAS.Eighty eligible patients initiated erlotinib therapy between March 2003 and May 2005. There were eight partial responses (10%), and an additional 33 patients (41%) had stable disease for 2 months or longer. The median TTP was 3.5 months (95% CI, 2.0 to 5.5 months). The median survival time was 10.9 months (95% CI, 7.8 to 14.6 months). The 1- and 2- year survival rates were 46% and 19%, respectively. The most common toxicities were acneiform rash (79%) and diarrhea (69%). Four patients developed interstitial lung disease of grade 3 or higher, with one treatment-related death. EGFR mutations were detected in nine of 43 patients studied. The presence of an EGFR mutation was strongly correlated with disease control, prolonged TTP, and survival.Erlotinib monotherapy is active and relatively well tolerated in chemotherapy-naïve elderly patients with advanced NSCLC. Erlotinib merits consideration for further investigation as a first-line therapeutic option in elderly patients.
Cyclin G, a recent addition to the cyclin family, was initially identified in screens for new srckinase family members and soon thereafter by differential screening for transcriptional targets of the tumor suppressor gene, p53. We have identified cyclin G as being overexpressed in breast and prostate cancer cells using differential display polymerase chain reaction screening. We demonstrate here that cyclin G is overexpressed in human breast and prostate cancer cells and in cancer cells in situ from tumor specimens. Cyclin G expression was tightly regulated throughout the cell cycle in normal breast cells, peaking at the S and G2/M phases of the cell cycle with lower levels in G1. The cell cycle-dependent expression was absent in breast cancer cells. Following DNA damage in normal p53+/+ cells, cyclin G is triggered to cluster in discrete nuclear DNA replication foci that contain replication-associated proteins such as proliferating cell nuclear antigen (PCNA). While p53−/− cells displayed a faint cyclin G nuclear staining pattern, there was no increased expression and no change in distribution of the staining pattern after DNA damage. The specific subcellular localization of cyclin G at DNA replication foci provides an additional link between p53-mediated growth arrest and cell cycle regulation and suggests that cyclin G may act as an effector of p53-mediated events by functional association with replication foci protein(s). Cyclin G, a recent addition to the cyclin family, was initially identified in screens for new srckinase family members and soon thereafter by differential screening for transcriptional targets of the tumor suppressor gene, p53. We have identified cyclin G as being overexpressed in breast and prostate cancer cells using differential display polymerase chain reaction screening. We demonstrate here that cyclin G is overexpressed in human breast and prostate cancer cells and in cancer cells in situ from tumor specimens. Cyclin G expression was tightly regulated throughout the cell cycle in normal breast cells, peaking at the S and G2/M phases of the cell cycle with lower levels in G1. The cell cycle-dependent expression was absent in breast cancer cells. Following DNA damage in normal p53+/+ cells, cyclin G is triggered to cluster in discrete nuclear DNA replication foci that contain replication-associated proteins such as proliferating cell nuclear antigen (PCNA). While p53−/− cells displayed a faint cyclin G nuclear staining pattern, there was no increased expression and no change in distribution of the staining pattern after DNA damage. The specific subcellular localization of cyclin G at DNA replication foci provides an additional link between p53-mediated growth arrest and cell cycle regulation and suggests that cyclin G may act as an effector of p53-mediated events by functional association with replication foci protein(s). Human cancer development is a multistage process that results from the stepwise acquisition of genetic alterations. These alterations may involve the dysregulation of a variety of normal cellular functions, leading to the initiation and progression of a tumor. Among normal cellular functions, regulatory control of the cell cycle plays an important role in normal cell proliferation, and genetic alterations that affect cell cycle control have been shown to be associated with tumor progression (reviewed in Refs. 1Pardee A.B. Science. 1989; 246: 603-608Crossref PubMed Scopus (1854) Google Scholar, 2Hunter T. Pines J. Cell. 1991; 66: 1071-1074Abstract Full Text PDF PubMed Scopus (387) Google Scholar, 3Hartwell L.H. Kastan M.B. Science. 1994; 266: 1821-1828Crossref PubMed Scopus (2316) Google Scholar). Cyclins are prime cell cycle regulators and control the major check points in cell cycle transitions of eukaryotic cells (2Hunter T. Pines J. Cell. 1991; 66: 1071-1074Abstract Full Text PDF PubMed Scopus (387) Google Scholar). In association with a family of cyclin-dependent protein kinases, cyclins maintain the orderly progression of cells through the various phases of the cell cycle. The link between oncogenesis and cyclins has been made with the aberrant expression of two cyclins (cyclin A and D1) in human tumors (2Hunter T. Pines J. Cell. 1991; 66: 1071-1074Abstract Full Text PDF PubMed Scopus (387) Google Scholar, 4Wang J. Chenivesse X. Henglein B. Brechot C. Nature. 1990; 343: 555-557Crossref PubMed Scopus (558) Google Scholar, 5Pines J. Hunter T. Nature. 1990; 346: 760-763Crossref PubMed Scopus (529) Google Scholar, 6Motokura T. Bloom T. Kim H.G. Juppner H. Ruderman J.V. Kronenberg H.M. Arnold A. Nature. 1991; 350: 512-515Crossref PubMed Scopus (1161) Google Scholar). Other members of the cyclins (i.e. cyclin B, D3, and E) have also been shown to have altered expressions in human tumors, including breast and prostate cancers (7Schuuring E. Veroeven E. Mooi W.J. Michalides R.J.A.M. Oncogene. 1991; 6: 439-444PubMed Google Scholar, 8Jiang W. Kahn S.M. Tomita N. Zhang Y.-J. Lu S.H. Weinstein B. Cancer Res. 1992; 52: 2980-2983PubMed Google Scholar, 9Keomarsi K. Pardee A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1112-1116Crossref PubMed Scopus (508) Google Scholar, 10Weinstar-Saslow D. Merino M.J. Manrow R.E. Lawrence J.A. Bluth R.F. Wittenbel K.D. Simpson J.F. Page D.L. Steeg P.S. Nat. Med. 1995; 1: 1257-1260Crossref PubMed Scopus (296) Google Scholar). Cyclin G (Cyc G), 1The abbreviations used are: Cyc G, cyclin G; NMEC, normal mammary epithelial cell; hNMEC, human normal mammary epithelial cell; mNMEC, mouse normal mammary epithelial cell; FACS, fluorescence-activated cell sorter; MMC, mitomycin C; PCR, polymerase chain reaction; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; ABC, avidin-biotin peroxidase complex; PBS, phosphate-buffered saline; PCNA, proliferating cell nuclear antigen. a recent addition to the cyclin family, was initially identified in screens for the src kinase family in rat fibroblasts and soon thereafter by differential screening for transcriptional targets of the tumor suppressor gene p53 (11Tamura K. Kanaoka Y. Jinno S. Nagata A. Ogiso Y. Shimizu K. Hayakawa T. Nojima H. Okayama H. Oncogene. 1993; 8: 2113-2118PubMed Google Scholar, 12Okamoto K. Beach D. EMBO J. 1994; 13: 4816-4822Crossref PubMed Scopus (455) Google Scholar). Cyc G has homology to fission yeast Cig1, B-type cyclins, and human cyclins A and I (13Horne M.C. Goolsby G.L. Donaldson K.L. Tran D. Neubauer M. Wahl A.F. J. Biol. Chem. 1996; 271: 6050-6061Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Unlike other cyclin family members, Cyc G lacks a "destruction box" motif that controls the ubiquitin-dependent degradation but contains an epidermal growth factor receptor-like autophosphorylation motif (11Tamura K. Kanaoka Y. Jinno S. Nagata A. Ogiso Y. Shimizu K. Hayakawa T. Nojima H. Okayama H. Oncogene. 1993; 8: 2113-2118PubMed Google Scholar, 13Horne M.C. Goolsby G.L. Donaldson K.L. Tran D. Neubauer M. Wahl A.F. J. Biol. Chem. 1996; 271: 6050-6061Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Cyc G is the only known cyclin that is transcriptionally activated by the p53 tumor suppressor gene, suggesting that it may play a role in p53-mediated cell growth control (12Okamoto K. Beach D. EMBO J. 1994; 13: 4816-4822Crossref PubMed Scopus (455) Google Scholar, 13Horne M.C. Goolsby G.L. Donaldson K.L. Tran D. Neubauer M. Wahl A.F. J. Biol. Chem. 1996; 271: 6050-6061Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 14Bates S. Rowan S. Vousden K.H. Oncogene. 1996; 13: 1103-1109PubMed Google Scholar, 15Smith M.L. Kontny H.U. Bortnick R. Fornace A.J. Exp. Cell Res. 1997; 230: 61-68Crossref PubMed Scopus (77) Google Scholar, 16Zauberman A. Lupo A. Oren M. Oncogene. 1994; 10: 2361-2366Google Scholar). Furthermore, despite its high homology with other cyclins, Cyc G has not yet been matched with a cyclin-dependent kinase binding partner, and its biological function remains elusive. However, Cyc G forms a complex with B′ regulatory subunits of protein phosphatase 2A following its induction by p53 (17Okamoto K. Kamibayashi C. Serrano M. Prives C. Mumby M.C. Beach D. Mol. Cell. Biol. 1996; 16: 6593-6602Crossref PubMed Scopus (88) Google Scholar). In contrast to most p53 target genes such asp21/Waf1/Cip1/Sdi1, Bax1, IGF-BP, andGadd45, Cyc G does not seem to exert a tumor-suppressive role but rather, like other cyclins or proto-oncogenes, plays a growth-promoting role (15Smith M.L. Kontny H.U. Bortnick R. Fornace A.J. Exp. Cell Res. 1997; 230: 61-68Crossref PubMed Scopus (77) Google Scholar, 18Wu L. Liu L. Yee A. Carbonaro-Hall D. Tolo V.T. Hall F. Oncol. Rep. 1994; 1: 705-711PubMed Google Scholar, 19Skotzko M. Wu L. Anderson W.F. Gordon E.M. Hall F.L. Cancer Res. 1995; 55: 5493-5498PubMed Google Scholar, 20Smith M.L. Bortnick R. Sheikh S. Fornace A.J. Exp. Cell Res. 1998; 242: 235-243Crossref PubMed Scopus (13) Google Scholar). Consistent with this observation, Cyc G overexpression has been observed in human osteosarcoma cells (18Wu L. Liu L. Yee A. Carbonaro-Hall D. Tolo V.T. Hall F. Oncol. Rep. 1994; 1: 705-711PubMed Google Scholar,19Skotzko M. Wu L. Anderson W.F. Gordon E.M. Hall F.L. Cancer Res. 1995; 55: 5493-5498PubMed Google Scholar). Initially, we identified the cyc G gene using differential screening (differential display PCR) (21Liang P. Pardee A.B. Science. 1992; 257: 967-971Crossref PubMed Scopus (4707) Google Scholar) between human normal and tumor breast cells. Here we report the expression pattern, cellular localization, cell cycle regulation, and Cyc G response to p53 induction in normal and breast cancer cells. We show that aberrant expression of Cyc G may be closely associated with the tumorigenic process. Our demonstration that following DNA damage, Cyc G is triggered to cluster in highly organized nuclear compartments representing DNA replication foci may provide an additional link between p53-mediated growth arrest and cell cycle control. Together, these data suggest that Cyc G may act as an effector in p53-mediated tumor suppression by functional association with replication foci protein(s). The quality of total RNA was tested by Northern blot analysis before reverse transcription. Differential display PCR was performed using the RNAimageTM Kit 1 according to the manufacturer's protocol (GenHunter). The cDNA products were amplified by the polymerase chain reaction using H-AP3 5′ primer (5′-AAGCTTTGGTCAG-3′) and H-T11G 3′ primer. The band of interest was isolated, PCR-reamplified using the same primers as above, cloned into pGEM-T vector (Promega), and sequenced by the dideoxynucleotide chain termination method with Sequenase (U.S. Biochemical Corp.). This partial-length clone was labeled with [α-32P]dCTP using the random primed synthesis method and used as a probe to obtain the full-length clone by screening a cDNA library prepared from normal mammary epithelial cells (22Lee S.W. Nat. Med. 1996; 2: 776-782Crossref PubMed Scopus (182) Google Scholar). Primary human normal mammary epithelial cells (hNMECs) were established from reduction mammoplasties obtained through the Cooperative Human Tissue Network and designated 14N and 15N, as described (23Emerman J.T. Wilkinson D.A. In Vitro Cell Dev. Biol. 1990; 26: 1186-1194Crossref PubMed Scopus (27) Google Scholar, 24Band V. Sager R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1249-1253Crossref PubMed Scopus (260) Google Scholar, 25Ethier S.P. Mahacek M.L. Gullick W.J. Frank T.S. Weber B.L. Cancer Res. 1993; 53: 627-635PubMed Google Scholar). Mouse mammary epithelial cells (mNMECs) were derived from 129/Sv (p53+/+) and 129/Sv-Trp53 m/Tyj (p53−/−) mice obtained from Jackson Laboratories. These cells were grown in DFCI-1 medium (D Complete) as described (24Band V. Sager R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1249-1253Crossref PubMed Scopus (260) Google Scholar) and were used at early to mid passage, i.e. 5–10 population doublings. Human breast cancer cells (MCF7, T47D, Hs578t, MDAMB435, and MDAMB436) were obtained from ATCC. Growth media for these mammary cancer cells was DMEM, 10% fetal bovine serum (FBS). Primary human normal prostate cells were derived from normal adjacent tumor tissue biopsies received from the Cooperative Human Tissue Network and designated NPrEC-1 and -2. These cultures were grown in Clonetics Prostate Epithelial Media. The human prostate carcinoma cell lines PC3, LNCaP, and DU145 were purchased from the ATCC and maintained in DMEM, 10% FBS. ND1 was kindly provided by Dr. P. Narayan. EJ-p53, EJ-CAT cells, and Saos 2-p53 cells were cultured in the presence or absence of tetracycline (1 μg/ml) in DMEM, 10% FBS as described previously (26Sugrue M.M. Shin D.Y. Lee S.W. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9648-9653Crossref PubMed Scopus (265) Google Scholar). To induce the expression of p53, cells were washed three times with PBS, and fresh medium without tetracycline was added. The murine cell lines Vm10, VhD, and 10.1 were a gift from Xiangwei Wu (Mount Sinai School of Medicine) and were cultured in DMEM, 10% FBS plus hygromycin (50 μg/ml) as described (27Chen J. Wu X. Lin J. Levine A.J. Mol. Cell. Biol. 1996; 16: 2445-2452Crossref PubMed Scopus (334) Google Scholar). hNMECs were synchronized by growth factor deprivation, lovastatin, or nacodazole block. Synchronization by growth factor deprivation has been described previously (28Keyomarsi K. Sandoval L. Band V. Pardee A.B. Cancer Res. 1991; 51: 3602-3609PubMed Google Scholar). Under these conditions, cells were arrested at G0 after the removal of growth factors from the growth media, as determined by [3H]thymidine incorporation. At time 0, cells were stimulated to re-enter the cell cycle by the addition of DFCI-1 complete medium with growth factors, and the DNA synthesis rate was estimated by measuring the incorporation of [3H]thymidine. Cells cycle synchronization in G1 was carried out using lovastatin as described (28Keyomarsi K. Sandoval L. Band V. Pardee A.B. Cancer Res. 1991; 51: 3602-3609PubMed Google Scholar). Briefly, hNMECs were grown in D complete medium supplemented with 15 μm lovastatin for 24 h. Cells were induced to enter the cell cycle by the removal of lovastatin and the addition of 1.5 mm mevalonic acid. Parallel cultures were collected at several time points to analyze mRNA and protein levels or were stained with propidium iodide and analyzed for DNA content using fluorescence-activated cell sorting (FACS) analysis. MCF7 cells were grown in DMEM, 10% FBS and were synchronized by 20 μmlovastatin for 33 h, followed by 2.0 mm mevalonic acid, and were harvested as described above. Nacodazole-induced G2/M-phase block was done as described (29Kiyokawa N. Yan D.-H. Brown M.E. Hung M.-C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1092-1096Crossref PubMed Scopus (30) Google Scholar). Briefly, cells were grown in medium supplemented with 50 ng/ml nacodazole for 24 h and then nacodazole was removed, cultures were washed, and fresh growth medium was added. Cells were harvested and analyzed as described above. For FACS analysis, cells were fixed with 80% ice-cold ethanol for 30 min and washed with PBS. Cells were suspended in RNase (10 μg/ml) and incubated at 37 °C for 30 min, washed, and stained for DNA content with 5 mg/ml propidium iodide. Cells were analyzed on a FACScan flow cytometer and analyzed with Elite software. Immunohistologic studies were performed on 4-μm frozen sections cut from tissues embedded in OCT. Tissue blocks were obtained from the Cooperative Human Tissue Network, eastern division. Immunostaining of sections was performed by the avidin-biotin peroxidase complex (ABC) method using the Vectastain Elite ABC kit (Vector Laboratories) as follows. Tissues were treated to remove endogenous peroxidase activity, blocked with goat serum for 30 min at room temperature, and then incubated in primary anti-Cyc G antibody (10 μg/ml; UBI) for 2 h. Sections were washed three times in PBS, 0.1% Tween 20 (PBST) and then incubated with a secondary anti-rabbit antibody conjugated to horseradish peroxidase for 30 min. Following PBS washes, the sections were incubated with ABC Elite reagent for 30 min at room temperature and washed three times in PBS. The bound horseradish peroxidase complexes were developed using diaminobenzidine tetrahydrochloride (Sigma Fastdab) according to the manufacturer's instructions. The sections were counterstained with hematoxylin, dehydrated, and mounted with glass coverslips. Cells were plated onto chamber slides (Lab-Tek), and when 50% confluent, they were treated with DNA-damaging agents as described below and then fixed with 100% methanol for 10 min at −20 °C, followed by permeabilization with 0.1% Triton X-100 for 5 min. Staining was carried out as follows. Cells were blocked with PBST containing 10% heat-inactivated goat serum, 3% nonfat dry milk for 30 min at room temperature. Primary antibodies (Cyc G; Santa Cruz Biotechnology Inc. (Santa Cruz, CA) catalog no. SC-320) or nonspecific rabbit IgG (10 μg/ml) was diluted with 0.1% nonfat milk/PBST. Cells were incubated for 1.5 h, rinsed in PBST, and then stained with a rhodamine-conjugated secondary antibody for 1 h. Cells were then observed with a Zeiss Axioscope I photomicroscope equipped with epifluorescence. Cyc G antibodies (UBI and Santa Cruz Biotechnology) and anti-PCNA (clone PC10; Oncogene) were used for dual labeling. Double immunofluorescence labeling was carried out with two different secondary antibodies specific to each primary antibody, and double exposures were taken. Co-localization between Cyc G (red, rhodamine) and PCNA (green, fluorescein isothiocyanate) proteins was tested on mitomycin C (MMC; 10 μg/ml)-treated hNMEC (15N) cells using two-color immunostaining. Detergent extraction of the cells followed by methanol fixation removed detergent-soluble PCNA and enabled the clear visualization of detergent-insoluble PCNA, the form that is tightly complexed to DNA in replication foci (23Emerman J.T. Wilkinson D.A. In Vitro Cell Dev. Biol. 1990; 26: 1186-1194Crossref PubMed Scopus (27) Google Scholar). Cells were grown to 50% confluence; MMC was added to cultures at 5 or 10 μg/ml; and cells were harvested after 6, 12, and 24 h of treatment. Actinomycin D was added to cultures at a final concentration of 10 or 20 ng/ml and harvested at 12, 24, and 48 h of treatment. Cells were also exposed to 6 Gy γ-irradiation in a Mark 137I cesium source irradiator and recovered for 12 h. Cells were collected in reducing SDS-polyacrylamide gel electrophoresis loading buffer and boiled for 5 min for Western blots or collected in guanidium isothiocyanate buffer for RNA extraction. For Western blot analysis, samples were adjusted for equal protein and separated on 12% SDS-polyacrylamide gels under reducing conditions. Gels were transferred to nitrocellulose membrane, and blots were probed with antibodies to Cyc G (UBI), Cdc2 (Santa Cruz Biotechnology; catalog no. 054), wild-type p53 (AB-6; Oncogene), and β-actin (clone AC-15; Sigma). Bands were detected using the ECL chemilluminescence detection method (Amersham Pharmacia Biotech) and exposed to x-ray film. For Northern blot analysis, total RNA was extracted, denatured, and electrophoresed through a 1% agarose-formaldehyde gel (20 μg of total RNA/lane). The gel was transferred to nylon membrane (Bio-Rad) and hybridized as follows. Human Cyc G, p21, p53, histone H4, and 36B4 probes were 32P-labeled by using random primed DNA labeling techniques. Blots were exposed on x-ray film after washing. In all cases, films were scanned (HP ScanJet IIcs) and analyzed using Adobe Photoshop software. Using the differential display PCR technique (21Liang P. Pardee A.B. Science. 1992; 257: 967-971Crossref PubMed Scopus (4707) Google Scholar) to survey differentially expressed clones in human mammary normal and tumor cells, we isolated a cDNA fragment, 15-1 clone, with higher abundance in cancer (T47D and MDAMB435) compared with normal cells (14N and 15N). After reamplification and subcloning, 15-1 cDNA fragment was used to confirm the differential display expression patterns on Northern blot. Sequence analysis and comparison of 15-1 clone to those within the GenBankTM data base revealed a 100% sequence match to the 3′-untranslated region of the human cyclin G cDNA (13Horne M.C. Goolsby G.L. Donaldson K.L. Tran D. Neubauer M. Wahl A.F. J. Biol. Chem. 1996; 271: 6050-6061Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 14Bates S. Rowan S. Vousden K.H. Oncogene. 1996; 13: 1103-1109PubMed Google Scholar). Using this partial clone as a probe, a normal human mammary cDNA library was screened and a full-length cDNA was isolated. We first examined the expression of Cyc G in human mammary normal and tumor epithelial cell lines. As shown in Fig.1, all human breast cancer cell lines examined had higher levels of Cyc G protein expression, regardless of estrogen receptor status, but a low level of expression was detected in human normal mammary epithelial cells (14N and 15N). In addition, higher levels of Cyc G protein were observed in human prostate tumor cell lines when compared with normal prostate epithelial cells (Fig.1). The distribution of Cyc G protein was determined using immunohistochemistry in a number of human normal and tumor breast tissue specimens to define the relationship between Cyc G expression and breast cancer development. A total of 40 different cases were analyzed, including 10 normal biopsies, and 30 tumor biopsies (10in situ carcinomas, seven infiltrating ductal carcinomas, six infiltrating lobular carcinomas, and seven fibroadenomas). Among the normal biopsies, little or no nuclear or cytoplasmic Cyc G staining was observed either in the epithelial or stromal cells of the ductal units (Fig. 2 A). However, 22 of the 30 tumor samples showed increased Cyc G expression that was specific to nuclei of epithelial cells (Fig. 2, C,E, and G). Positive nuclear staining was observed in seven of the 10 in situ carcinomas, five of six infiltrating ductal carcinomas, four of six infiltrating lobular carcinomas, and six of seven fibroadenomas. With the exception of the fibroadenomas, stromal cells of the tumor biopsies showed no staining. Our findings indicate that Cyc G overexpression is a frequent occurrence in breast cancer. To gain further insight into the possible function(s) of Cyc G, we compared the expression patterns of Cyc G mRNA and protein throughout the cell cycle in normal versus tumor cells (Fig.3, A and B). A normal human breast epithelial cell strain (15N) and a human mammary cancer cell line (MCF7) were synchronized by three different methods: 1) lovastatin (cells arrest at G1); 2) growth factor deprivation (G0; normal cells only); and 3) nacodazole (G2/M). Synchronization of both cell types was monitored by [3H]thymidine incorporation, histone H4 expression, and flow cytometry analysis (FACS) (Fig. 3 C). Northern and Western blot analyses of cells harvested at regular intervals revealed a differential expression pattern of Cyc G during cell cycle progression in normal versus tumor cells (Fig. 3). In normal cells (NMECs), Cyc G mRNA and protein levels were low to undetectable during most of G1 phase. As cells entered into S phase, a marked induction of Cyc G was observed, which remained high throughout the S and G2/M phases, correlating with histone H4 expression (Fig. 3 A). A similar pattern of expression was observed with all three synchronization methods in normal cells. However, in a human breast cancer line, MCF7, cell cycle-dependent expression was absent; instead, there was a consistent high level of expression throughout the cell cycle (Fig.3 B). MCF7 cells were synchronized by lovastatin or nacodazole, both of which gave similar patterns of Cyc G expression (Fig. 3 B). Cyc G overexpression in cancer cells may result from an altered cell cycle regulation. cyc Gis a transcriptional target gene of p53 that contains two p53-binding elements in its promotor region (12Okamoto K. Beach D. EMBO J. 1994; 13: 4816-4822Crossref PubMed Scopus (455) Google Scholar, 16Zauberman A. Lupo A. Oren M. Oncogene. 1994; 10: 2361-2366Google Scholar). High basal levels of Cyc G expression in tumor cells lacking functional p53 suggest that altered expression of Cyc G may be independent of p53 dysregulation and may contribute to the malignant phenotype of cancer cells. To examine the possibility of an altered response of Cyc G by p53 in some cell types, we have utilized a tetracycline-regulated p53-inducible system in tumor cell lines (Saos2-p53 and EJ-p53) that contain nonfunctional p53 and a high basal level of Cyc G. As shown in Fig.4 A, after tetracycline removal, p53 mRNA was readily detectable (data not shown) as well as p21 mRNA, a p53 transcriptional target gene. After removal of tetracycline in both cell lines, Cyc G levels remained unchanged, although p21 mRNA was induced dramatically after 12 h, indicating p53 transcriptional activity. This level of expression remained consistent and unchanged over time (up to 5 days), suggesting no specific induction of Cyc G expression by p53 in these cell types. However, the expression of Cyc G was dramatically induced following wild-type p53 activation in VhD and Vm10 cells (27Chen J. Wu X. Lin J. Levine A.J. Mol. Cell. Biol. 1996; 16: 2445-2452Crossref PubMed Scopus (334) Google Scholar), which express low basal levels of Cyc G (Fig. 4 B). The VhD and Vm10 are immortalized mouse embryo fibroblast cell lines containing a temperature-sensitive p53 mutant (Val to Ala mutation at codon 135, tsp53), which express a nonfunctional p53 at 37–39 °C and fully functional p53 at 32 °C. The VhD cells undergo reversible G1 arrest in a p53-dependent manner at 32 °C, while the Vm10 cell line, which constitutively expresses tsp53 and the c-myc oncogene undergoes apoptosis at 32 °C (27Chen J. Wu X. Lin J. Levine A.J. Mol. Cell. Biol. 1996; 16: 2445-2452Crossref PubMed Scopus (334) Google Scholar). The expression of p21 in these two tsp53 cell lines was low before the temperature shift and increased with similar kinetics to Cyc G induction. Altogether, these data demonstrate that the activation of Cyc G expression by p53 induction or activation is universal for both growth arrest and apoptotic pathways but only observed in cells containing low basal expression of Cyc G. In order to determine regulation and localization of Cyc G in response to DNA damage, we first examined the expression and localization of Cyc G in mNMECs isolated from p53-deficient mice after treatment with DNA damaging agents. Cells from the mammary glands of p53 null mice as well as cells from wild type mice were treated with actinomycin D (not shown) or MMC. Northern blot analysis showed that Cyc G mRNA expression was induced after treatment with actinomycin D in wild-type cells, while in p53 null cells, Cyc G mRNA was hardly detectable, and no induction was observed after DNA damage treatment (Fig. 5 A,top). We also examined the cells for distribution of Cyc G protein before and after DNA damage with MMC using immunofluorescence staining (Fig. 5 A, bottom). In wild-type cells, the staining with anti-Cyc G antibodies revealed an amplification of distinct punctate patterns in nuclei following DNA damage treatment, while untreated cells had a lower number of punctate nuclear signals. These MMC-treated wild-type cells also showed strong recruitment of p53 to the nuclei. However, p53 null cells displayed a very faint Cyc G nuclear staining pattern, and there was no increase of signals after DNA damage (Fig. 5 A). We next compared the distribution of Cyc G before and after DNA damage in human normal and tumor mammary epithelial cells (Fig.5 B). When 15N cells (hNMEC) were methanol-fixed and stained for Cyc G, a distinct but low level nuclear signal was detected (Fig.5 B), similar to that seen in p53+/+ mNMEC (Fig.5 A). The staining correlated well with the Western blot analysis, which showed low levels of Cyc G protein in NMECs compared with tumor cell lines (Fig. 1). After exposure to MMC, a dramatic amplification of Cyc G signal was observed, which was localized to the nucleus in a discrete staining pattern, consisting of 6–10 brightly labeled clusters per cell reminiscent of DNA replication foci (Fig.5 B). In human mammary cancer cells, the patterns of Cyc G localization were not as distinct as those seen in normal cells, although the staining was still present in the nucleus (Fig.5 B). We used two tumor mammary cancer cell lines: MCF7, containing wild-type p53, and T47D, containing a nonfunctional mutated p53 gene (30Wosikowski K. Regis J.T. Robey R.W. Alvarez M. Buters J.T. Gudas J.M. Bates S.E. Cell Growth Differ. 1995; 6: 1395-1403PubMed Google Scholar). Both lines have higher basal expression levels of Cyc G as compared with hNMEC (Fig.5 B). MCF7 cells displayed amplified nuclear staining signals in response to DNA damage, but T47D cells did not show any induction of Cyc G protein by DNA damage (Fig. 5 B). The distribution of staining for both cell types also appeared predominantly nuclear. The localization observed after DNA damage in normal cells suggests that Cyc G may be recruited to areas of DNA replication and repair, delineated as DNA replication foci, where proteins such as PCNA and several cyclins including cyclin A and D have been shown to colocalize (31Xiong Y. Zhang H. Beach D. Cell. 1992; 71: 505-514Abstract Full Text PDF PubMed Scopus (902) Google Scholar). Given the pattern of Cyc G localization in the nucleus and the potential involvement of Cyc G in DNA replication and repair, we explored the possibility of an association between Cyc G and PCNA, which plays an essential role in nucleic acid metabolism as a component of the replication and repair machinery. Co-localization between Cyc G and PCNA proteins was tested
Initiation of the Epstein-Barr virus (EBV) lytic cycle is dependent on expression of the viral transactivator Zta, which is encoded by the BZLF1 gene. Described here is an initial mapping of the regions of Zta involved in activating transcription. The data indicate that the amino-terminal 153 amino acids of Zta are important for activity, and in particular the region from residues 28 to 78 appears to be critical for Zta function. However, other features of Zta may be important for activity since a Gal4-Zta chimeric protein, generated by fusing the amino-terminal 167 residues of Zta to the DNA binding domain of the yeast transactivator Gal4, transactivated a minimal promoter containing one upstream Gal4 binding site but was unable to exhibit synergistic transactivation when assayed with a reporter containing five upstream Gal4 binding sites.
Serum response factor (SRF), a member of the MCM1, agamous, deficiens, SRF (MADS) family of transcriptional activators, has been implicated in the transcriptional control of a number of cardiac muscle genes, including cardiac α-actin, skeletal α-actin, α-myosin heavy chain (α-MHC), and β-MHC. To better understand the in vivo role of SRF in regulating genes responsible for maintenance of cardiac function, we sought to test the hypothesis that increased cardiac-specific SRF expression might be associated with altered cardiac morphology and function. We generated transgenic mice with cardiac-specific overexpression of the human SRF gene. The transgenic mice developed cardiomyopathy and exhibited increased heart weight-to-body weight ratio, increased heart weight, and four-chamber dilation. Histological examination revealed cardiomyocyte hypertrophy, collagen deposition, and interstitial fibrosis. SRF overexpression altered the expression of SRF-regulated genes and resulted in cardiac muscle dysfunction. Our results demonstrate that sustained overexpression of SRF, in the absence of other stimuli, is sufficient to induce cardiac change and suggest that SRF is likely to be one of the downstream effectors of the signaling pathways involved in mediating cardiac hypertrophy.
EGFR is frequently mutated and amplified in lung adenocarcinomas sensitive to EGFR inhibitors gefitinib and erlotinib. A secondary mutation, T790M, has been associated with acquired resistance but has not been shown to be sufficient to render EGFR mutant/amplified lung cancers resistant to EGFR inhibitors. We created a model for studying acquired resistance to gefitinib by prolonged exposure of a gefitinib-sensitive lung carcinoma cell line (H3255; EGFR mutated and amplified) to gefitinib in vitro. The resulting resistant cell line acquired a T790M mutation in a small fraction of the amplified alleles that was undetected by direct sequencing and identified only by a highly sensitive HPLC-based technique. In gefitinib-sensitive lung cancer cells with EGFR mutations and amplifications, exogenous introduction of EGFR T790M effectively conferred resistance to gefitinib and continued ErbB-3/PI3K/Akt signaling when in cis to an activating mutation. Moreover, continued activation of PI3K signaling by the PIK3CA oncogenic mutant, p110alpha E545K, was sufficient to abrogate gefitinib-induced apoptosis. These findings suggest that allelic dilution of biologically significant resistance mutations may go undetected by direct sequencing in cancers with amplified oncogenes and that restoration of PI3K activation via either a T790M mutation or other mechanisms can provide resistance to gefitinib.
Abstract Purpose: Mutations in the epidermal growth factor receptor (EGFR) are associated with clinical and radiographic responses to EGFR tyrosine kinase inhibitors gefitinib and erlotinib. Currently available methods of EGFR mutation detection rely on direct DNA sequencing, which requires isolation of DNA from a relatively pure population of tumor cells, cannot be done on small diagnostic specimens, and lack sensitivity. Here we describe the use of a sensitive screening method that overcomes many of these limitations. Experimental Design: We screened 178 non–small cell lung cancer specimens for mutations in exons 18 to 21 of EGFR using a DNA endonuclease, SURVEYOR, which cleaves mismatched heteroduplexed DNA. Samples were analyzed by high-performance liquid chromatography on the Transgenomic WAVE HS system. Selected specimens that produced digestion products using SURVEYOR were subsequently reanalyzed by size separation or under partially denaturing conditions, followed by fractionation and sequencing. The specimens included DNA isolated from frozen tumor specimens, dissected formalin-fixed, paraffin-embedded tumor specimens undergoing clinical sequencing, and undissected formalin-fixed, paraffin-embedded specimens. One hundred sixty specimens were independently analyzed using direct DNA sequencing in a blinded fashion. Results: EGFR mutations were detected in 16 of 61 fresh frozen tumor specimens, 24 of 91 dissected formalin-fixed, paraffin-embedded tumor specimens, and 11 of 26 undissected formalin-fixed, paraffin-embedded tumor specimens. Compared with sequencing, the sensitivity and specificity of the present method were 100% and 87%. The positive and negative predictive values were 74% and 100%, respectively. SURVEYOR analysis detected 7 (4%) mutations that were not previously detected by direct sequencing. Conclusions: SURVEYOR analysis provides a rapid method for EGFR mutation screening with 100% sensitivity and negative predictive value. This unbiased scanning technique is superior to direct sequencing when used with undissected formalin-fixed, paraffin-embedded specimens.
