Greater enhancement of chemosensitivity by caffeine in high-p185neu-expressing human non-small-cell lung cancer cell lines.
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Biomarkers, Tumor↗
Biomedical subjects
M C Hung
Publications and source records attributed to M C Hung.
c-erbB-2/neu overexpression enhances metastatic potential of human lung cancer cells by induction of metastasis-associated properties.
Previously, we and others have reported high levels of expression of the c-erbB-2/neu gene in non-small cell lung cancer cell lines and primary tumors. We have also found that expression of c-erbB-2/neu-encoded p185neu was correlated with lymph node metastasis in lung squamous cell carcinomas. To investigate the potential role of the c-erbB-2/neu gene in lung cancer metastasis systematically, we introduced the human c-erbB-2/neu gene into very low p185neu-expressing NCI-H460 human non-small cell lung cancer cells and then examined the experimental metastatic potentials among the parental NCI-H460 cells and stable transfectants with increased expression of p185neu. Compared with the parental NCI-H460 cells, the NCI-H460 transfectants overexpressing p185neu produced significantly more pulmonary and extrapulmonary metastatic tumors in nude mice. The changes in experimental metastatic potential in vivo were accompanied by increased invasiveness in vitro. In addition, important steps in the invasion and metastasis process, such as secretion of basement membrane-degradative enzymes and migration through reconstituted basement membrane (Matrigel), were also increased in the NCI-H460 transfectants overexpressing p185neu. Moreover, scanning electron microscopy revealed that the p185neu-overexpressing NCI-H460 transfectants had significantly more microvilli and membrane protrusions than the parental cells, correlating with the increased invasive properties of these cells. The results demonstrate that overexpression of p185neu can enhance the experimental metastatic potential of NCI-H460 human lung cancer cells by promoting invasion and the other steps in the metastatic cascade.
Animals↗
Hyperthermia enhances the cytotoxicity of National Institutes of Health 3T3 cells transfected with a noncleavable transmembrane pro-tumor necrosis factor deletion mutant.
Hyperthermia has been shown to potentiate the cytotoxicity of exogenously added tumor necrosis factor (TNF) against tumor cell targets. The mechanism for that interaction is not known, but among the possibilities are that heat enhances internalization of ligand-bound TNF or enhances processing of internalized TNF. In this study, we found that NIH 3T3 cells transfected with an expression vector containing the full-length human pro-TNF secreted TNF and that hyperthermic treatment of chromium-labeled L929 target cells at 43 degrees C for 1 h potentiated the cytotoxicity of these transfectants against the L929 cells in clonogenic survival and chromium-release assays. On the other hand, transfectants expressing a transmembrane, nonsecretable pro-TNF mutant that kills L929 cells by cell-to-cell contact without internalization also exhibited enhanced cytotoxicity against heated L929 cell targets. Thus, potentiation of the cytotoxicity of TNF by hyperthermia is not strictly dependent on enhanced internalization of ligand-bound TNF or enhanced processing of internalized TNF.
3T3 Cells↗
The retinoblastoma gene product, Rb, represses neu expression through two regions within the neu regulatory sequence.
The neu oncogene is frequently overexpressed in breast, ovarian and lung cancers, and its overexpression correlates with poor disease prognosis. The exact mechanism of deregulation of neu expression is not well understood. Our previous studies indicate that the tumor suppressor retinoblastoma gene product, Rb, represses transcription of neu through the GTG enhancer (-243 to -234 relative to initiation of translation site of rat neu). We carried out further deletion analysis of the regulatory sequences of neu and found that Rb also represses neu close to transcription initiation sites (-172 to -79). Bal 31 deletions downstream of nucleotide -172 show that the sequence TCGAGGAA (-172 to -165) is important for efficient transcription from the neu promoter and also for repression by Rb. Rb mutants with mutations in the large T/E1a binding domain repress transcription from transcription initiation sites but not the GTG enhancer, suggesting that Rb modulates different regions of the regulatory sequence of neu by different pathways. The net effect of the Rb mutants is to repress not only transcription but also the transforming activity of activated neu in focus-forming assays. Thus, one mechanism whereby Rb may act as a tumor suppressor is to repress transcription of the strongly transforming neu oncogene.
3T3 Cells↗
TNF cytotoxicity: effects of HER-2/neu expression and inhibitors of ADP-ribosylation.
Previous studies have implicated ADP-ribosylation in the mechanism of TNF cytotoxicity. In short-term 51Cr-release assays with several mouse and human tumor cell lines, the inhibitors aminobenzamide (ABA) and nicotinamide (NA) of ADP-ribosylation sensitized HER-2/neu-nonoverexpressing cells (CaOV-3 and MCF-7) but not HER-2/neu-overexpressing cells (SKOV-3 and SKBR-3) to TNF. However, both inhibitors alone or in combination with either TNF and/or actinomycin D (AD) caused similar effects on ADP-ribosylation rates of CaOV-3 and SKOV-3 cells after 4 h of treatment. This result suggests that ADP-ribosylation may not be involved in sensitizing these human tumor cells to TNF. Both ABA and NA decreased the TNF sensitivity of L929 cells and either increased or decreased TNF sensitivity of EMT-6 cells in the absence or presence of actinomycin D, respectively. Again, there was no correlation between ADP-ribosylation and TNF cytotoxicity in these mouse cell lines. Thus, modulation of TNF sensitivity by these inhibitors might be linked to a compromised repair mechanism distinct from the effects on ADP-ribosylation alone.
Adenosine Diphosphate Ribose↗
Transcriptional regulation of neu by RB and E1A in rat-1 cells.
Functional inactivation of the tumor-suppressing retinoblastoma gene (Rb) is involved in the etiology of many types of human cancers, including hereditary retinoblastomas. The neu gene is a dominant transforming oncogene, and we previously found that the Rb-encoded protein (RB) suppresses neu-induced transformation in NIH3T3 cells by repressing transcription of the neu oncogene. We report here that RB was unable to repress neu oncogene transcription in Rat-1 cells but could functionally antagonize transcriptional repression of neu by the adenovirus E1A. Mutant forms of RB that have mutations in either the E1A-binding or carboxy-terminal regions had less or no antagonizing effects on E1A-mediated repression of neu in Rat-1 cells. Results of focus-formation assays showed that the transformation activity of the neu oncogene in Rat-1 cells could be regulated by E1A and RB in accordance with their transcriptional regulation activities. The data demonstrate that RB can regulate transcription of neu in a negative or positive manner depending on the cell type. Carboxy terminus of RB as well as the E1A-binding region can mediate transcriptional regulation. Based on these results, we propose a model for the complex transcriptional regulation of neu by RB and E1A.
Adenoviridae↗
Reexpression of neu-encoded oncoprotein counteracts the tumor-suppressing but not the metastasis-suppressing function of E1A.
By transfecting the adenovirus 5 E1A gene into neu-transformed NIH3T3 cells, we previously showed that E1A can dramatically repress neu-encoded p185 expression and, concomitantly, suppress the features of transformation and metastasis of neu+E1A transfectants. From these results we concluded that suppression of transformation and metastasis by E1A in neu-transformed cells may be through repression of neu gene expression. However, E1A has recently been shown to also repress the transformation features of other human cancer cells that do not overexpress neu. This observation raised a possibility that repression of neu gene expression in our neu+E1A cells might not be the only mechanism for transformation and metastasis suppression. To study whether other molecular mechanisms might be involved in suppression of transformation and metastasis by E1A in our neu+E1A cells, we reexpressed p185 oncoprotein in the neu+E1A cells by transfecting them with a plasmid containing activated rat neu complementary DNA and we examined whether E1A can suppress transformation and metastasis when the neu-encoded p185 protein is reexpressed. All the features of transformed cells including cell morphology, DNA synthesis rate, colony formation in soft agar, and tumorigenicity in nu/nu mice were restored in the cell lines that reexpressed neu. In addition, the levels of neu reexpression corresponded to the degree of malignant transformation. However, the in vivo metastatic tumor formation by these p185 reexpressing cells was still significantly inhibited by E1A. When metastasis-associated properties were examined in the cell lines that reexpressed p185, we found that cell motility was recovered by reexpression of p185 to the degree corresponding to the p185 reexpression level, but secretion of membrane-degrading gelatinases and invasion through the basement membrane preparation Matrigel by these cells were still inhibited by E1A. The data demonstrated that reexpression of p185 in neu+E1A cells can counteract the tumor-suppressing function of E1A but not completely recover the neu-induced metastatic phenotype. We conclude from these results that (a) repression of neu oncogene expression was indeed the molecular mechanism by which E1A suppressed tumor formation in neu-transformed 3T3 cells, and (b) suppression of metastasis by E1A in neu-transformed 3T3 cells was via multiple molecular mechanisms in addition to repressing neu. Our model system clearly demonstrated that tumorigenicity and metastasis are related but separable phenomena.
3T3 Cells↗
Downstream signal transduction defects that suppress transformation in two revertant cell lines expressing activated rat neu oncogene.
The neu gene encodes the transmembrane tyrosine kinase growth factor receptor, p185. To study neu-induced cellular transformation, we developed revertant cells from the neu-transformed NIH 3T3 cell line, B104-1-1, by treating the cells with the chemical mutagen ethyl methanesulfonate. The morphologically normal revertant cells were first selected by their ability either to attach to culture plates or to survive in the presence of the cytotoxic reagents colchicine or 5-fluoro-2-deoxyuridine. Two of the 21 candidate revertant cell lines isolated were further characterized and were found to lose their anchorage independence and ability to grow in 1% calf serum, indicating that they were nontransformed even though they still expressed p185 oncoprotein. The tyrosine residues of p185 in these two revertants were underphosphorylated, which may have contributed to their nontransformed status. In addition, these revertants also resisted transformation by neu and several additional oncogenes (H-ras, N-ras, v-mos, v-abl, and v-fos) as determined by focus forming assays. These results indicated that we had successfully developed, from neu-transformed cells, revertants exhibiting defective tyrosine phosphorylation of the neu oncoprotein. The results also suggested that neu and several other oncogenes may share common elements in their pathways for the induction of cellular transformation.
3T3 Cells↗
Effects of truncation of human pro-tumor necrosis factor transmembrane domain on cellular targeting.
Human tumor necrosis factor is initially synthesized as a transmembrane prohormone anchored by a hydrophobic region of the leader sequence. This hydrophobic domain has been previously localized to extend from Leu-46 to Ile-21 based on hydropathy calculations. To functionally determine the nature of this domain, we have generated a series of pro-TNF mutant cDNAs in which either half or both halves of this encoding domain is deleted. These cDNAs were analyzed both by the ability of their mRNAs to direct translation in a microsomal system and by cellular localization of their encoded TNFs following transfection of NIH/3T3 cells. We determined that the mutant protein with deletion of the periluminal region of the transmembrane domain (Thr-32 to Ile-21) was translocated into microsomes and localized on the inner surface of the microsomal membrane in a fashion identical to that of the parental TNF. In contrast, the mutants with deletion of either the pericytoplasmic aspect (Leu-46 to Gly-34) or of virtually the entire transmembrane domain were not localized in the microsomes. Transfection experiments indicated that only the cDNAs whose peptide products were translocated across microsomal membranes gave rise to transmembrane prohormones and matured TNFs. Thus, the functions of membrane targeting and orientation prior to proteolytic processing can be fulfilled by the sequence Leu-46 to Ala-33 of the transmembrane domain, but not by the sequence Ala-33 to Ile-21.
Amino Acid Sequence↗
Enhanced c-erbB-2/neu expression in human ovarian cancer cells correlates with more severe malignancy that can be suppressed by E1A.
Amplification or overexpression of c-erbB-2/neu protooncogene, or both, occur frequently in many different types of human cancers and have been shown to correlate with decreased survival in ovarian cancer patients. We have previously found that the ovarian carcinoma cell line SK-OV-3 overexpresses c-erbB-2/neu mRNA. To further study the biological effect of c-erbB-2/neu overexpression in SK-OV-3 cells, we injected such cells i.p. into female nu/nu mice and found that this cell line forms extensive abdominal tumors and ascites. From the ascites in an injected mouse, we established the SKOV3.ip1 cell line and found that it expressed 2-fold more c-erbB-2/neu-encoded p185 proteins than the parental SK-OV-3 cells. When transformation phenotypes of SK-OV-3 and SKOV3.ip1 cells were compared, SKOV3.ip1 cells showed higher cell growth and DNA synthesis rates, formed more colonies in soft agar, produced larger s.c. tumors, and resulted in shorter survival of nu/nu mice after i.p. injection. These data indicate that the level of c-erbB-2/neu overexpression may correlate with the degree of malignancy in these ovarian carcinoma cells. Since we had previously shown that the adenovirus 5 E1A gene product can suppress transformation and metastatic properties induced by mutation-activated rat neu oncogene in mouse embryo fibroblast cells, we further examined whether E1A can abrogate malignancy in c-erbB-2/neu-overexpressing human ovarian cancer cells. We introduced the E1A gene into c-erbB-2/neu-overexpressing SKOV3.ip1 cells and found that the E1A-expressing ovarian cancer cell lines had decreased c-erbB-2/neu-encoded p185 expression and reduced malignancy, including a decreased ability to induce tumors in nu/nu mice. Therefore, we concluded that E1A is a tumor suppressor gene for c-erbB-2/neu-overexpressing human ovarian cancer cells and may be useful in developing therapeutic reagents for these human cancers.
Adenovirus E1A Proteins↗
Repression of neu-induced clonogenicity by dimethylsulfoxide correlates with decreased levels of neu-encoded cell-surface p185 and changes in phosphotyrosine content of endogenous proteins.
Treatment of neu-transformed fibroblasts with dimethylsulfoxide (DMSO), results in change in morphology and loss of clonogenicity. Although the total amount of neu-encoded p185 protein and mRNA remained constant after DMSO treatment, cell-surface p185 decreased by 60%, indicating that transmembrane p185 protein is not located in its physiological position. The aberrant location of p185 induced by DMSO resulted in increased tyrosine phosphorylation of p185 and concomitant decreased tyrosine phosphorylation of potential substrate proteins of p185 in the cell. However, the autophosphorylation activity of p185 in vitro was unaffected by DMSO. Thus, DMSO-induced loss of clonogenicity may be due to inappropriate location of p185, which prevents interaction between p185 and its substrates and therefore inhibits p185-mediated signal transduction pathway.
Animals↗
Differential activity of the RVF enhancer element in the decreased expression of the neu oncogene in NR-6 cells versus parental Swiss Webster 3T3 cells.
The rat neu oncogene encodes a growth factor receptor-like glycoprotein, termed p185, that shares structural similarity with epidermal growth factor receptor (EGFR), particularly in the tyrosine-kinase domain. The Swiss Webster 3T3 murine embryo fibroblast (SW3T3) variant NR-6, in contrast to the parental cell line, does not express EGFR mRNA. After transfection of an activated rat neu cosmid clone, we demonstrated in this report that whereas SW3T3 cells readily expressed the exogenous rat p185 protein, NR-6 cells did not express detectable levels of this protein product. By transfection of plasmids containing the chloramphenicol acetyltransferase (CAT) gene driven by the neu promoter and subsequent CAT assays, we also showed that neu gene promoter activity was significantly less in NR-6 cells than in SW3T3 cells and that the neu promoter sequence responsible for this decreased transcription was a previously identified RVF enhancer element (Yan D-H, Hung M-C, Mol Cell Biol 11:1875-1882, 1991). That is to say, the RVF enhancer element of the neu promoter did not function as an enhancer in NR-6 cells. To investigate the mechanism responsible for the inactivation of RVF in NR-6 cells, we used southwestern blot analyses and demonstrated that the 60-kDa RVF polypeptide was present in both NR-6 and SW3T3 cell nuclear extracts. This result indicates that the DNA-binding activity of RVF was similar in these two cell lines; therefore, loss of RVF enhancer activity in NR-6 cells is probably due to inactivation of the trans-activating function and not DNA binding activity of RVF.
3T3 Cells↗
Negative regulation of the neu promoter by the SV40 large T antigen.
The neu gene is amplified and its protein product is overexpressed in certain human tumors. The adenovirus 5 E1a gene product and c-myc repress neu transcription. Moreover, expression of E1a in neu-transformed cells leads to decrease in transformation phenotype and metastatic potential. The simian virus 40 large T antigen (LT) shares structural and functional homology with E1a and c-myc, and all three proteins bind to the retinoblastoma gene product, Rb. We found that LT also represses neu expression at the transcriptional level. However, LT represses neu promoter by a different mechanism compared to E1a and c-myc, because the region of the neu promoter mediating repression by LT (-172 to -79) is downstream from the region responding to E1a and c-myc (-312 to -172). In addition, a LT mutant (K1) unable to complex Rb still represses neu promoter activity, indicating that the Rb binding domain of LT is not required for repression of neu. Since K1, unlike LT, does not transform Rat-1 cells but, like LT, represses the neu promoter, we tested whether K1 functions as a transformation suppressor of activated neu oncogene. Focus-forming assays showed that K1 indeed suppresses the strong cell-transforming activity of activated neu.
Animals↗
Epidermal growth factor receptor expression and function control cytotoxic responsiveness to tumor necrosis factor in ME-180 squamous carcinoma cells.
Tumor necrosis factor (TNF) induces dose-dependent but incomplete cytotoxicity in ME-180 cervical carcinoma cells, suggesting that TNF response heterogeneity exists within this cell line. To investigate cellular properties associated with TNF-mediated cytotoxicity, ME-180 cell variants were isolated that stably expressed complete resistance (ME-180R) or sensitivity (ME-180S) to TNF and were compared to the parental cell line. Analysis of 125I-TNF binding on variant and parental cells provided evidence for postreceptor regulation of TNF responsiveness. Epidermal growth factor (EGF) receptor expression in TNF-resistant ME-180R cells was 3-fold higher than that expressed in the parental population and 4-fold higher when compared to TNF-sensitive ME-180S cells. High expression levels correlated with increased EGF receptor tyrosine kinase activity and receptor tyrosine phosphorylation. Southern and Northern blot analysis provided evidence of amplification and overexpression of the EGF receptor gene and its message in ME-180R cells which approached the content and alternate mRNA species expressed in A431 cells. Although 3-fold greater levels of total cellular EGF receptor protein were detected in ME-180R cell lysates, and its cell surface localization was confirmed by immunodetection, these cells were paradoxically unable to bind greater quantities of 125I-EGF or to express greater cytotoxic sensitivity to an EGF-diphtheria toxin fusion protein. Overall, these results suggest that expression of EGF receptor protein, its intrinsic tyrosine kinase activity, or its phosphotyrosine content may alter TNF cytotoxic signal transduction and control TNF responsiveness within the ME-180 cell line.(ABSTRACT TRUNCATED AT 250 WORDS)
Carcinoma, Squamous Cell↗
Transcriptional repression of the neu protooncogene by estrogen stimulated estrogen receptor.
Both neu gene overexpression and loss of estrogen receptor (ER) expression have been found to correlate with a poor prognosis in human breast cancer. Studies of breast tumor specimens have suggested that these two factors are not independent, leading us to hypothesize that there is a causal relationship between loss of ER and overexpression of neu. In this report, we confirm that ER can negatively regulate the expression of the neu gene protein product, p185neu, in two ER positive but not an ER negative breast cancer cell line(s). We have produced sublines which stably express human ER from a previously ER negative human breast cancer cell line. We demonstrate that the expression of ER in these cell lines is sufficient to confer the ability to respond to estradiol by down-regulating neu expression at both the protein and RNA levels. Utilizing neu promoter-chloramphenicol acetyltransferase constructs in transient cotransfection assays, we have also shown that this regulation occurs at the transcriptional level and requires the presence of both ER and estradiol. Furthermore, utilizing promoter deletion constructs, we provide evidence that a 140-base pair region of the neu promoter is required for this transcriptional regulation. When placed in a heterologous promoter, this 140-base pair region allows transcriptional repression by estradiol stimulated ER; thus, it represents an estrogen responsive region within the neu promoter. Finally, we have used gel mobility shift analysis to demonstrate an alteration in the nuclear factor(s) binding to this promoter region in estradiol stimulated versus estradiol deprived breast cancer cells. This study provides the first evidence that the inverse clinical correlation between neu and ER expression may be due to transcriptional repression of neu by estradiol stimulated ER.
Down-Regulation↗
Oral cancer progression and c-erbB-2/neu proto-oncogene expression.
Monoclonal antibody PAb3 to c-erbB-2/neu protein was utilized in the immunoperoxidase staining of 86 human specimens from oral mucosa. These tissue specimens represented a spectrum from 7 normal to 9 simple hyperplasia, 15 mild dysplasia, 14 moderate dysplasia, 20 severe dysplasia and 21 squamous cell carcinoma. Our study indicated that as the cells acquire a more malignant phenotype, there was a progressive increase in neu expression. It also suggested that neu may be involved in the development of oral cancers and that its evaluation in the early stages may assist in the diagnosis and management of oral cancers.
Carcinoma, Squamous Cell↗
Differential expression of the neu oncogene in mouse liver and pancreatic cell lines.
To study the tissue-specific expression of neu oncogene, we used two mouse tumor cell lines derived from liver, Hep1-a, and pancreatic, 266-6, tumors as a model system. The endogenous neu gene is expressed in Hep1-a but not in 266-6 cells. We demonstrate in this report that differential expression of the neu gene in these two cell lines is mainly regulated at transcriptional level. The neu promoter sequence responsible for the differential regulation is localized within a 90 bp region and it is possibly due to lack of a specific positive transcription factor(s) interacting with this region in 266-6 cells.
Animals↗
The retinoblastoma gene product suppresses neu oncogene-induced transformation via transcriptional repression of neu.
The retinoblastoma susceptibility gene (Rb) is a tumor suppressor gene involved in the etiology of many types of human cancers. However, the molecular mechanisms involved in tumor suppression by Rb are largely unknown. The neu gene is a dominant transforming oncogene and a member of the growth factor receptor tyrosine kinase gene family. Both inactivation of the Rb gene and overexpression of the neu gene are involved in human breast and lung cancers. Therefore, it is of interest and importance to investigate the potential interactions between Rb and neu. Here we show that Rb suppresses neu-induced transformation by focus formation assays. This transformation suppression by Rb was further shown to be due to transcriptional repression of neu using Rb expressing effector plasmid and neu promoter-chloramphenicol acetyltransferase reporter gene. The cis-acting element conferring Rb-mediated repression was mapped to a recently identified novel enhancer in the neu promoter. The data indicate that the growth factor receptor neu is a target for the Rb gene product and transcriptional repression of a dominant oncogene expression may be one of the molecular mechanisms of Rb-mediated tumor suppression.
3T3 Cells↗