scholarly journals MBRS-42. YB-1 - A NOVEL THERAPEUTIC TARGET IN HIGH-RISK MEDULLOBLASTOMA?

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii405-iii405
Author(s):  
Louisa Taylor ◽  
Ian Kerr ◽  
Beth Coyle
Keyword(s):  
Cell Lines ◽  
Nuclear Translocation ◽  
Genomic Analysis ◽  
Tissue Microarrays ◽  
Nuclear Accumulation ◽  
Pcr Analysis ◽  
Elevated Expression ◽  
Group 3 ◽  
Chemotherapeutic Treatment ◽  
Ccaat Box

Abstract Medulloblastoma relapse occurs in 30–40% of patients and is typically fatal. The emergence of therapy resistant sub-clones likely plays a major role in a large proportion of recurrent medulloblastoma. Y-box binding protein 1 (YB-1) is a multifunctional transcription/translation factor and known onco-protein. Overexpression has been described in numerous cancers, where elevated expression and nuclear accumulation correlates with disease progression, metastasis and drug resistance. Genomic analysis of a large medulloblastoma cohort revealed YB-1 up-regulation across all subgroups of medulloblastoma, where elevated expression correlated with poor survival. Immunohistochemical staining of patient tissue microarrays displayed significant YB-1 expression, with a high proportion (83%) of patients exhibiting nuclear accumulation. High YB-1 expression was also observed at both protein and RNA level across medulloblastoma cell lines, with expression highest in Group 3 and 4. Hence, we hypothesised that YB-1 plays a role in medulloblastoma chemoresistance and progression. Treatment of Group 3 (HDMB-03 and D283MED) and SHH (DAOY) cell lines with vincristine and cisplatin and analysis of cellular localisation by nuclear/cytoplasmic fractionation and immunofluorescence demonstrated that YB-1 undergoes nuclear translocation in response to these standard medulloblastoma chemotherapy agents. Chromatin immunoprecipitation (ChIP) analysis of untreated Group 3 cell lines (D283MED and HDMB-03) demonstrated considerable YB-1 interaction with an inverted CCAAT box in the ATP-binding cassette subfamily B member 1 (ABCB1) promoter. RT-PCR analysis of ABCB1 following vincristine and cisplatin treatment revealed differences in transcript expression, indicative of different YB-1 promoter interactions dependent on chemotherapeutic treatment. Our results highlight YB-1 as a novel candidate chemoresistance driver in medulloblastoma.

scholarly journals Knockdown of CAVEOLIN-1 Sensitizes Human Basal-Like Triple-Negative Breast Cancer Cells to Radiation

2017 ◽  
Vol 44 (2) ◽  
pp. 778-791 ◽  
Author(s):  
Man Zou ◽  
Yanhui Li ◽  
Shu Xia ◽  
Qian Chu ◽  
Xiaoguang Xiao ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Dna Repair ◽  
Cell Lines ◽  
Triple Negative ◽  
Nuclear Translocation ◽  
Nuclear Accumulation ◽  
Caveolin 1 ◽  
Tnbc Cell ◽  
Radiation Induced ◽  
Her 2

Background/Aims: Triple-negative breast cancer (TNBC) is a high-risk breast cancer phenotype without specific targeted therapy options and is significantly associated with increased local recurrence in patients treated with radiotherapy. CAVEOLIN-1 (CAV-1)-mediated epidermal growth factor receptor (EGFR) nuclear translocation following irradiation promotes DNA repair and thus induces radiation resistance. In this study, we aimed to determine whether knockdown of CAV-1 enhances the radiosensitivity of basal-like TNBC cell lines and to explore the possible mechanisms. Methods: Western blotting was used to compare protein expression in a panel of breast cancer cell lines. Nuclear accumulation of EGFR as well as DNA repair and damage at multiple time points following irradiation with or without CAV-1 siRNA pretreatment were investigated using western blotting and confocal microscopy. The radiosensitizing effect of CAV-1 siRNA was evaluated using a clonogenic assay. Flowcytometry was performed to analyse cell apoptosis and cell cycle alteration. Results: We found that CAV-1 is over-expressed in basal-like TNBC cell lines and barely expressed in HER-2-positive cells; additionally, we observed that HER-2-positive cell lines are more sensitive to irradiation than basal-like TNBC cells. Our findings revealed that radiation-induced EGFR nuclear translocation was impaired by knockdown of CAV-1. In parallel, radiation-induced elevation of DNA repair proteins was also hampered by pretreatment with CAV-1 siRNA before irradiation. Silencing of CAV-1 also promoted DNA damage 24 h after irradiation. Colony formation assays verified that cells could be radiosensitized after knockdown of CAV-1. Furthermore, G2/M cell cycle arrest and apoptosis enhancement may also contribute to the radiosensitizing effect of CAV-1 siRNA. Conclusion: Our results support the hypothesis that CAV-1 knockdown by siRNA causes increased radiosensitivity in basal-like TNBC cells. The mechanisms associated with this effect are reduced DNA repair through delayed CAV-1-associated EGFR nuclear accumulation and induction of G2/M arrest and apoptosis through the combined effects of CAV-1 siRNA and radiation.

Preferential Nuclear Accumulation of JAK2V617F In CD34+ but Not Granulocytic, Megakaryocytic or Erythroid Cells of Patients with Philadelphia-Negative Myeloproliferative Neoplasia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4089-4089
Author(s):  
Ciro R Rinaldi ◽  
Paola Rinaldi ◽  
Adele Alagia ◽  
Marica Gemei ◽  
Vitalyi Senyuk ◽  
...  
Keyword(s):  
Cell Lines ◽  
Nuclear Localization ◽  
Nuclear Translocation ◽  
Nuclear Accumulation ◽  
Erythroid Cells ◽  
Jak2 Inhibitor ◽  
Preferential Accumulation ◽  
Cd34 Cells ◽  
V617f Mutation ◽  
Nuclear Signal

Abstract Abstract 4089 Constitutive activation of JAK2 by chromosomal translocations or a point mutation is a frequent event in hematological malignancies particularly in Philadelphia-negative MPN. Recently, Dawson et al. identified a novel nuclear role of JAK2 in the phosphorylation of Tyr 41 of histone H3 leading to chromatin displacement of HP1a in hematopoietic cell lines and in the CD34+ cells collected from the peripheral blood of one PMF patient with JAK2V617F mutation. To determine whether the V617F mutation observed in MPN patients affects the sub-cellular localization of JAK2, we first analyzed by confocal immunofluorescence (CIF) microscopy and Western Blot (WB), K562 cells stably transfected with pMSCV-puroJAK2V617F or pMSCV-puroJAK2. The results confirm the nuclear and cytoplasmic localization of JAK2 in K562 as reported by Dawson et al. However, we consistently observed a much stronger nuclear signal in the cells expressing JAK2V617F than in those carrying wt JAK2 suggesting that the mutation leads to a preferential accumulation of JAK2V617F in the nucleus. To determine whether there is a preferential nuclear translocation of JAK2V617F in vivo, we analyzed by CIF microscopy and WB the total BM cells of 10 JAK2V617F-positive MPN patients (ET n=4, PV n=3, PMF n=3, allele burden median: 56%, 70%, 72% respectively) and of 5 MPN patients with wt JAK2 (PMF n= 2, ET n=3). We found a strong nuclear signal in mononucleated cells of 10 of 10 JAK2V617F-positive patients but not in those with wt JAK2. The JAK2 signal was observed almost exclusively in the nucleus suggesting a predominantly nuclear homing of JAK2V617F. To identify the phenotype of these cells, we used fluorescence activated cell sorting (FACS) to isolate CD34+, CD15+, CD41+ and CD71+ fractions from the BM of three JAK2V617F-positive MPN patients (1 ET, 1 PV, 1 early PMF). We found nuclear JAK2 in CD34+ positive cells collected from BM only in V617F mutated patients. No obvious nuclear signal was detected in differentiated granulocytic, megakaryocytic and erythroid cells obtained from the patients (n=15). To determine whether the block of JAK2 activity could interfere with nuclear localization of JAK2, we incubated JAK2V617F and JAK2 expressing K562 with the selective JAK2 inhibitor AG490. At the IC50 dose (25 uM) and after 3 h of incubation, CIF images showed the JAK2 redistribution in the vast majority of V617F expressing K562 and the replacement in the cytoplasm but not in wt cells. By QRT-PCR we demonstrated that the V617F mutation strongly up-regulates LMO2 expression in K562 and in CD34+ cells. In our assay, the addiction of AG490 progressively and completely restore LMO2 levels in V617F expressing K562. Our data corroborate recently published results of a nuclear localization of JAK2 in hematopoietic cells and they also extend these findings by showing that in all subtypes of MPN patients JAK2V617F accumulates in the nucleus of progenitor CD34+ cells while remains mostly in the cytoplasm of their differentiated progeny. The chromatin alterations due to the preferential accumulation of JAK2V617F in the nucleus correlates with a significant increase in LMO2 expression in cell lines and in sorted CD34+ cells. The selective JAK2 inhibitor AG490 is able to revert nuclear JAK2 and normalize LMO2 levels in vitro, suggesting how the block in JAK2 nuclear translocation could be a new treatment strategy for JAK2 mutated patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals MBRS-45. TWIST1 AND ABCB1 ARE FUNCTIONAL DETERMINANTS OF METASTASIS IN MEDULLOBLASTOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii406-iii406
Author(s):  
Alice Cardall ◽  
Franziska Linke ◽  
Ian Kerr ◽  
Beth Coyle
Keyword(s):  
Cell Lines ◽  
Metastatic Disease ◽  
Epithelial Mesenchymal Transition ◽  
Efflux Pump ◽  
Migration And Invasion ◽  
Pcr Analysis ◽  
Mesenchymal Transition ◽  
Chip Sequencing ◽  
Metastatic Cell ◽  
Group 3

Abstract Paediatric medulloblastomas (MB) are frequently metastatic, resulting in a poor prognosis for the patient. Of the four MB subgroups, group 3 patients present with the highest rates of metastasis and worst outcomes. The mechanisms behind the metastatic process are poorly understood, limiting our ability to develop novel therapeutic treatments. We hypothesised that the epithelial-mesenchymal transition (EMT) transcription factor TWIST1 and the multidrug efflux pump ABCB1 (ATP-binding cassette subfamily B member 1) synergistically drive MB metastasis. TWIST1 protein expression was analysed in patient tissue microarrays by immunohistochemistry. High TWIST1 expression was associated with metastatic patients (p=0.041). Physical and functional interactions between TWIST1 and ABCB1 were investigated using chromatin immunoprecipitation (ChIP) and a 3D migration and invasion model. ChIP analysis confirmed TWIST1 binding to the ABCB1 promoter in SHH (ONS-76) and group 3 (D283MED and HD-MB03) metastatic cell lines. TWIST1 and ABCB1 were inhibited in HDMB03 cells with harmine and vardenafil respectively, resulting in attenuated cell migration in the 3D model. Western blot and qRT-PCR analysis of harmine treated cells confirmed a reduction in ABCB1 protein and gene expression. Overall our data reveals TWIST1 and ABCB1 to be key targets for MB metastatic disease. Using bioinformatics analysis and ChIP sequencing, additional TWIST1 downstream targets are now being identified and compared across the metastatic cell lines (ONS-76, D283MED and HD-MB03). This data will provide a deeper insight into the pathways associated with MB metastases, enabling personalised treatment approaches for patients with metastatic disease.

Effects of Elevated BCR/ABL Expression Level on Cellular Transformation and Sensitivity to Imatinib in Primitive Human Hematopoietic Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1998-1998
Author(s):  
Hardik Modi ◽  
Su Chu ◽  
Tinisha McDonald ◽  
Stephen Forman ◽  
Ravi Bhatia
Keyword(s):  
Cell Lines ◽  
Kinase Inhibitor ◽  
Cell Transformation ◽  
Hematopoietic Cells ◽  
Fold Increase ◽  
Cellular Transformation ◽  
Pcr Analysis ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Elevated Expression

Abstract Increased levels of BCR/ABL (BA) expression in CML hematopoietic cells have been associated with disease progression and resistance to the tyrosine kinase inhibitor imatinib mesylate (IM). Although cell lines with varying levels of BA expression have been studied, the role of elevated BA expression in cell transformation and drug resistance has not been directly evaluated in the primitive human hematopoietic cells in which the disease arises. Here we have used a human transduction model of CML to determine the effects of varying BA expression levels on cellular transformation (proliferation, apoptosis and differentiation) as well as responsiveness to IM. Cord blood (CB) CD34+ cells were transduced with MSCV vectors expressing BA and GFP, or control vectors expressing GFP alone followed by CD34+GFP+ cells selection by flow cytometry sorting. For BA expressing cells, two separate populations were selected based on low or high GFP expression (BAlo and BAhi). Quantitative RT-PCR analysis confirmed increased expression of BA in high GFP expressing cells (5.9±1.5 fold increase in BA:B2M levels in BAhi compared with the BAlo cells, n=3). The proliferation rate of BAhi cells, measured by fold expansion after 3 days of growth factor (GF) culture, was 2.0±0.2 fold higher than BAlo cells, and 6.7±0.1 fold higher than control (n=3). Upon GF deprivation, BAhi cells demonstrated increased resistance to apoptosis (24.4±11.4%, n=3) compared with BAlo (42±12%, n=3) or control cells (45±12%, n=3). BA transduced CD34+ cells generated higher numbers of glycophorin A+ cells than control (35.6%) following GF culture for 7 days. This effect was enhanced in BAhi (91.5%) compared with BAlo cells (77.1%). This was accompanied by an increase in CD33+ myeloid cells and a decrease in CD11b+ cells (36.3, 60.1 and 65.2% CD33+ cells and 6.0, 1.8 and 0.5% CD11b+ cells for control, BAlo and BAhi cells respectively). In addition the frequency of CD41a+ megakaryocytic cells was higher in BAhi (7.0%) relative to BAlo (3.1%) and control cells (1.0%). Next, we asked whether elevated expression of BA resulted in altered sensitivity to IM (0.025μM to 1μM) in an MTS assay. We observed that BAhi cells were more sensitive (83% inhibition at 1μM) to the IM compared with BAlo cells (14% inhibition at 1μM). We also investigated whether elevated levels of expression of two BA kinase mutants, M351T and E255K, were associated with altered IM sensitivity in CD34+ cells. The M351T mutation leads to intermediate level of IM resistance in cell lines. As was observed for wild type BA, CD34+ cells expressing higher levels of M351T demonstrated increased sensitivity to IM. On the other hand, cells expressing high and low levels of the E255K mutant, which is associated with high levels of IM resistance, demonstrated similar levels of IM sensitivity. In conclusion, increased levels of BA expression in human CD34+ cells results in enhanced proliferation; increased resistance to apoptosis following GF withdrawal, and altered differentiation with increased expression of erythroid, megakaryocytic and early myeloid markers and reduced expression of mature myeloid markers. Interestingly, expression of high levels of BA was associated with enhanced rather than reduced sensitivity to IM. Taken together these observations suggest that the effects of varying levels of BA expression on imatinib sensitivity in primitive human hematopoietic cells are determined primarily by increased proliferation rather than reduced apoptosis resulting from enhanced BA expression.

DDRE-37. YB-1 AS A BIOMARKER FOR DRUG RESISTANCE AND TUMOUR PROGRESSION IN MEDULLOBLASTOMA

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi82-vi82
Author(s):  
Louisa Taylor ◽  
Ian Kerr ◽  
Beth Coyle
Keyword(s):  
Drug Resistance ◽  
Cell Lines ◽  
Large Scale ◽  
Tumour Progression ◽  
Genomic Analysis ◽  
Immunohistochemical Analysis ◽  
Therapy Resistance ◽  
Cellular Stress Response ◽  
Nuclear Accumulation ◽  
Poor Overall Survival

Abstract Medulloblastoma (MB) relapse is the most significant unmet clinical challenge in childhood cancer. Recently it has become evident that MBs display altered biology at relapse, indicative of the emergence and expansion of a minor, therapy resistant cancer cell population. Thus, the examination of mechanisms underlying therapy resistance is of critical importance. Y-box binding protein 1 (YB-1) is a multi-functional oncoprotein whose elevated expression and nuclear accumulation correlate with drug resistance, metastasis and disease progression in numerous cancers, although little is known about the functional role of YB-1 in MB. Genomic analysis of large-scale publicly available patient datasets revealed YB-1 expression is significantly elevated across MB molecular subgroups and high expression correlates with poor overall survival. Immunohistochemical analysis of YB-1 localisation in patient TMAs revealed significant YB-1 nuclear accumulation, suggestive of elevated YB-1 nuclear activity in these patients. Treatment of Group 3 MB cell lines (D283MED and HDMB-03) with cisplatin and subsequent analysis by nuclear/cytoplasmic fractionation and confocal microscopy revealed significantly increased nuclear and overall YB-1 expression, indicating a role for YB-1 in cellular stress response. In support of this, ChIP analysis in D283MED and HDMB-03 cell lines confirmed YB-1 interaction with multi-drug transporter gene ABCB1, while stable YB-1 knockdown resulted in significantly reduced ABCB1 expression. Likewise, knockdown of YB-1 expression in D283MED cells results in increased susceptibility of cells to vincristine, supporting a role for YB-1 in the acquisition of drug resistance in MB cell lines. Finally, whole transcriptome sequencing of YB-1-knockdown HDMB-03 and D283MED cell lines indicated YB-1 regulation of a variety of cell death, survival and metabolic pathways. We are currently using ChIP-Seq analysis to identify targetable YB-1 downstream “hits” which drive these processes. Ultimately, we aim to identify druggable targets of YB-1 allowing us to establish more effective therapeutic options for the treatment of high-risk MB.

scholarly journals DNase II mediates a parthanatos-like developmental cell death pathway in Drosophila primordial germ cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Lama Tarayrah-Ibraheim ◽  
Elital Chass Maurice ◽  
Guy Hadary ◽  
Sharon Ben-Hur ◽  
Alina Kolpakova ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Germ Cells ◽  
Nuclear Translocation ◽  
Primordial Germ Cells ◽  
Nuclear Accumulation ◽  
Dependent Manner ◽  
Dnase Ii ◽  
Cell Death Pathway ◽  
Parp 1

AbstractDuring Drosophila embryonic development, cell death eliminates 30% of the primordial germ cells (PGCs). Inhibiting apoptosis does not prevent PGC death, suggesting a divergence from the conventional apoptotic program. Here, we demonstrate that PGCs normally activate an intrinsic alternative cell death (ACD) pathway mediated by DNase II release from lysosomes, leading to nuclear translocation and subsequent DNA double-strand breaks (DSBs). DSBs activate the DNA damage-sensing enzyme, Poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) and the ATR/Chk1 branch of the DNA damage response. PARP-1 and DNase II engage in a positive feedback amplification loop mediated by the release of PAR polymers from the nucleus and the nuclear accumulation of DNase II in an AIF- and CypA-dependent manner, ultimately resulting in PGC death. Given the anatomical and molecular similarities with an ACD pathway called parthanatos, these findings reveal a parthanatos-like cell death pathway active during Drosophila development.

scholarly journals Ruxolitinib Combined with Gemcitabine against Cholangiocarcinoma Growth via the JAK2/STAT1/3/ALDH1A3 Pathway

Biomedicines ◽  
2021 ◽  
Vol 9 (8) ◽  
pp. 885
Author(s):  
Shin-Yi Chung ◽  
Yi-Ping Hung ◽  
Yi-Ru Pan ◽  
Yu-Chan Chang ◽  
Chiao-En Wu ◽  
...  
Keyword(s):  
Cell Lines ◽  
Nuclear Translocation ◽  
Vital Role ◽  
First Line ◽  
Current Standard ◽  
Integrated Network ◽  
Jak2 Inhibitor ◽  
Therapeutic Drugs ◽  
Cholangiocarcinoma Cell ◽  
Malignant Behavior

Cholangiocarcinoma is the most common primary malignant tumor of the bile duct. The current standard first-line treatment for advanced or metastatic cholangiocarcinoma is gemcitabine and cisplatin. However, few effective treatment choices exist for refractory cholangiocarcinoma, and additional therapeutic drugs are urgently required. Our previous work demonstrated that the ALDH isoform 1A3 plays a vital role in the malignant behavior of cholangiocarcinoma and may serve as a new therapeutic target. In this study, we found a positive correlation between ALDH1A3 protein expression levels and the cell migration abilities of three cholangiocarcinoma cell lines, which was verified using ALDH1A3-overexpressing and ALDH1A3-knockdown clones. We also used ALDH1A3-high and ALDH1A3-low populations of cholangiocarcinoma cell lines from the library of integrated network-based cellular signatures (LINCS) program and assessed the effects of ruxolitinib, a commercially available JAK2 inhibitor. Ruxolitinib had a higher cytotoxic effect when combined with gemcitabine. Furthermore, the nuclear translocation STAT1 and STAT3 heterodimers were markedly diminished by ruxolitinib treatment, possibly resulting in decreased ALDH1A3 activation. Notably, ruxolitinib alone or combined with gemcitabine led to significantly reduced tumor size and weight. Collectively, our studies suggest that ruxolitinib might suppress the ALDH1A3 activation through the JAK2/STAT1/3 pathway in cholangiocarcinoma, and trials should be undertaken to evaluate its efficacy in clinical therapy.

scholarly journals Elevated Expression of the Testis-specific Gene WBP2NL in Breast Cancer

2015 ◽  
Vol 7 ◽  
pp. BIC.S19079 ◽  
Author(s):  
Seyedmehdi Nourashrafeddin ◽  
Mehdi Dianatpour ◽  
Mahmoud Aarabi ◽  
Maryam Beigom Mobasheri ◽  
Golnesa Kazemi-oula ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Line ◽  
Cell Lines ◽  
Specific Gene ◽  
Rt Pcr ◽  
Noncancerous Tissue ◽  
Ww Domain ◽  
Group I ◽  
Elevated Expression ◽  
Cancer Tissues

Breast cancer is one of the most common causes of cancer death in women; therefore, the study of molecular aspects of breast cancer for finding new biomarkers is important. Recent studies have shown that WW domain-binding protein 2 (WBP2) is important for the oncogenic property of breast cancer. WWP2 N-terminal-like ( WBP2NL) is a testis-specific signaling protein that induces meiotic resumption and oocyte activation events. Our previous study revealed that WBP2NL gene expression is elevated in actively dividing cells and it might be associated with cellular proliferation and tumorigenic process. However, the clinical relevance and importance of WBP2NL gene in cancer has not been understood yet. Therefore, we were interested in analyzing the expression of WBP2NL gene in human breast cancer tissues and breast cancer cell lines, for the first time. We used reverse transcription-polymerase chain reaction (RT-PCR) and semi-nested RT-PCR to evaluate the expression of WBP2NL in malignant breast cancer and adjacent noncancerous tissue (ANCT) samples, as well as MCF-7 and MDA-MB-231 cell lines. The WBP2NL gene was expressed in 45 out of 50 (90%) breast cancer tissues and overexpressed in the MDA-MB-231 cell line. We suggest that WBP2NL may play roles in breast cancer activation maybe through binding to a group I WW domain protein. The elevated expression of WBP2NL gene in breast cancer and MDA-MB-231 cell line leads us to suggest that WBP2NL might be considered as a novel prognostic factor for early diagnosis of breast cancer.

Deregulation of c-myc gene expression in human colon carcinoma is not accompanied by amplification or rearrangement of the gene

1985 ◽  
Vol 5 (8) ◽  
pp. 1969-1976
Author(s):  
M D Erisman ◽  
P G Rothberg ◽  
R E Diehl ◽  
C C Morse ◽  
J M Spandorfer ◽  
...  
Keyword(s):  
Colon Carcinoma ◽  
Cell Lines ◽  
Blot Hybridization ◽  
Human Colon ◽  
Normal Colonic Mucosa ◽  
Dna Rearrangement ◽  
Dot Blot ◽  
Myc Oncogene ◽  
Elevated Expression ◽  
High Level

The structure and expression of the c-myc oncogene were examined in 29 primary human colon adenocarcinomas. Dot blot hybridization of total RNA showed that 21 tumors (72%) had considerably elevated expression of c-myc (5- to 40-fold) relative to normal colonic mucosa. These data were corroborated by Northern blots of polyadenylated RNA, which showed a 2.3-kilobase transcript. Southern analysis of the c-myc locus in these tumors indicated the absence of amplification or DNA rearrangement in a 35-kilobase region encompassing the gene. In a parallel study, elevated expression of c-myc without amplification or DNA rearrangement was also observed in three of six colon carcinoma cell lines examined; in addition, unlike a normal colon cell line control, these three cell lines exhibited constitutive, high-level expression of the gene during their growth in cultures. These results indicate that elevated expression of the c-myc oncogene occurs frequently in primary human colon carcinomas and that the mechanism involved in the regulation of c-myc expression is altered in tumor-derived cell lines.

Sign in / Sign up

Export Citation Format

Share Document