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To determine whether normal breast cells have different levels of activating protein 1 (AP-1) expression and activation relative to breast cancer cells, we have compared the level of c-Jun and c-Fos expression and AP-1 activity in human mammary epithelial cells (HMECs) at different stages of transformation (normal proliferating HMECs, immortal HMECs, oncogene-transformed HMECs, and breast cancer cell lines). These studies demonstrated that normal and immortal HMECs have a high basal level of expression of cJun and cFos and higher AP-1 DNA-binding and transcriptional activating activities than do oncogene-transformed HMECs or human breast cancer cells, with a gradual decrease in AP-1 transactivating activity as cells progress through the carcinogenesis pathway (normal > immortal > oncogene-transformed > cancer cell lines). The AP-1 activity in normal or immortal cells was not modulated by growth factor supplementation or oncogene overexpression, as it is in breast cancer cells. However, the addition of suramin, a nonspecific growth factor antagonist, did inhibit AP-1 in these HMECs, suggesting that this high level of AP-1 present in normal HMECs may be due to autocrine stimulation of growth factor pathways. The differences in AP-1 activity in normal and malignant breast cells may indicate that normal cells are more dependent on AP-1-mediated signals for their growth than are breast cancer cells.

The purpose of this study was to document induction of apoptosis by vitamin E succinate (VES; RRR-alpha-tocopheryl succinate) in human breast cancer cells in culture and to characterize potential c-jun involvement. VES at 18.8 microM (10 micrograms/mL) induced DNA synthesis arrest, reduced total cell numbers, and induced apoptosis in estrogen receptor-positive and estrogen-responsive MCF-7 human breast cancer cells. VES at 10 micrograms/mL induced apoptosis in greater than 60% of cells within 3 d of treatment. Apoptosis was documented by detection of fragmented or condensed nuclei in 4',6-diamindino-2-phenylindole-stained cells, detection of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeled DNA, and DNA laddering. Analyses of mRNA and protein levels of candidate molecules involved in apoptosis showed that MCF-7 cells treated with VES exhibited elevated and persistent expression of c-jun. MCF-7 cells stably transfected with a dominant-negative interfering mutant c-jun, TAM-67, and expressing high levels of mutant jun exhibited approximately 50% blockage of VES-mediated apoptosis. In addition to increased c-jun expression after VES treatment, VES-treated MCF-7 cells exhibited elevated activator protein-1 (AP-1) binding activity. Comparisons of AP-1 binding factors by super-shift analyses with jun-specific antibodies in cells sensitive to VES-induced apoptosis (empty-vector control 7-1 cells) and cells resistant to VES-induced apoptosis (TAM-67-containing TAM-9 cells) showed that the sensitive cells expressed c-jun and jun D and the resistant cells TAM-67 AP-1 binding proteins after VES treatment. These studies suggested that c-jun may be involved in the apoptotic process initiated by VES treatment of human MCF-7 breast cancer cells.

The fragile histidine triad (FHIT) gene, located at 3p14.2, has been shown to be altered in numerous epithelial cancers. Because previous studies have shown a loss of heterozygosity and cytogenetic abnormalities at the 3p region in ovarian, endometrial, and cervical carcinomas, we examined the status of the FHIT gene in 14 ovarian, 8 cervical, and 4 endometrial human cancer cell lines. RNA was isolated and subjected to reverse transcription-PCR to amplify the FHIT gene transcript. Sixty-three % (5 of 8) of cervical cell lines, 14% (2 of 14) of ovarian cell lines, and none (0 of 4) of the endometrial cell lines displayed aberrantly migrating FHIT transcripts. DNA sequencing demonstrated that the aberrantly migrating bands primarily lacked exons 5, 6, and 7 (with other exon losses also observed), resulting in shorter mRNA transcripts. Southern blot analysis of DNA from five of the cervical carcinomas demonstrated alterations in four of them, three of which had exhibited no normally sized FHIT transcripts. The results suggest that the expression of the FHIT gene may be altered in cervical tumor tissue, potentially implicating this gene in cervical tumorigenesis, whereas the involvement of this gene appears to be less important in the development of ovarian and endometrial cancer.

Uterine sarcomas are unusual neoplasms of the female genital tract whose molecular etiology is largely unknown. We examined 20 leiomyomas as well as 23 uterine leiomyosarcomas for the presence of mutations in the Ki-ras and p53 genes, and overexpression of the MDM2 gene. Codons 12, 13, and 61 from the Ki-ras gene were characterized for the presence of mutations by restriction enzyme polymorphisms using mismatched primers and nucleic acid sequencing as appropriate. Activated Ki-ras genes were identified in 3/20 leiomyomas and 0/23 leiomyosarcomas. The p53 gene was analyzed by SSCP, nucleic acid sequencing, and immunohistochemical staining. None of 20 leiomyomas and 6/23 leiomyosarcomas exhibited p53 abnormalities. The SSCP/sequencing results did not consistently correlate with the IHC staining. MDM2 overexpression occurred in 0/20 leiomyomas and 3/23 sarcomas. Clinical correlation suggested that tumors with p53 mutations have a higher histologic grade or stage at presentation. We conclude that leiomyomas and leiomyosarcomas have different patterns of molecular alterations and are separate biologic entities. In addition, p53 and MDM2 overexpression may play a role in the development of a subset of leiomyosarcomas.

In an attempt to characterize the molecular alterations of cervical adenocarcinoma, we analyzed 32 paraffin-embedded specimens for the presence of K-ras mutations, p53 overexpression, p16 and Rb protein expression, and the presence of HPV 16 and 18 DNA. Overall 25/32 (78%) of the tumors displayed an abnormality in at least one of these analyses. K-ras mutations were detected by PCR amplification and RFLP analysis in 3 tumors, including 2 at codon 12 and 1 at codon 61. p53 overexpression determined by immunohistochemistry was demonstrated with > 80% of tumor nuclei staining in 4 cases, 10-15% of nuclei staining in 3 cases, and < 1% of nuclei staining in 5 cases. The pattern of staining was diffuse in 6 cases, focal in 1 case, and scattered in 5 cases. Analysis of p16 protein expression in 23 specimens revealed 1 tumor with abnormal staining, while Rb protein expression was determined to be normal in all 25 tumors tested. HPV DNA, detected by PCR with type-specific primers, was found in 16 tumors (50%), including 7 (22%) with HPV 16 and 9 (28%) with HPV 18. There was no correlation among these abnormalities except that the presence of HPV and strong p53 overexpression (> 80% tumor nuclei staining) were mutually exclusive events. Clinical correlation demonstrated that p53 overexpression involving the majority of tumor cell nuclei is characteristic of advanced stage disease, while HPV positivity and activated ras genes are associated with early stage disease.

Cells respond to external signals by either activating or inhibiting key regulatory proteins of gene expression called transcription factors (TFs). Abnormal expression of these factors plays a critical role in many human cancers. Recently, many TFs have been identified and their structure-function relationship has been characterized. Such new information has been useful in designing new chemotherapeutic drugs targeting these regulatory proteins. This review discusses the use of dominant-negative (DN) mutants of TFs as inhibitors of cell growth, as well as possible strategies for screening potential inhibitors of TF activity.

We have previously described a dominant-negative mutant of cJun that lacks the transactivation domain (TAD) of cJun and prevents AP-1-mediated transcriptional activation by quenching endogenous Jun or Fos proteins. We now report the development of a panel of cJun mutants that have inactivating mutations in the TAD, DNA-binding domain (DBD), or leucine zipper domain. These mutants are all unable to activate transcription, but only TAD and DBD mutants function in a dominant-negative fashion by inhibiting both cJun-induced transcriptional activation and transformation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate in ras-transfected rat embryo cells. Although the TAD and DBD mutants both function as transdominant inhibitors, they work through different mechanisms and with different inhibitory potencies. The DBD mutants, which function by inhibiting DNA binding, are relatively weak inhibitors, whereas the TAD mutants inhibit by quenching and are much more potent. Dimerization assays demonstrate that mutations in the DBD decrease the dimerization affinity of these mutants with cJun. These results demonstrate that the most potent dominant-negative mutants of cJun are proteins that have intact DBDs and quench the activity of the endogenous transcription factors.

The proto-oncogene c-jun encodes a component of the AP-1 transcription-activating complex and has been implicated in the regulation of diverse cellular processes, including cell proliferation, differentiation, transformation, and most recently, apoptosis. We have used a U937 monocytic leukemia cell line stably expressing a c-jun dominant-negative, transactivation-domain deletion mutant (TAM67) to assess the role of c-jun in apoptotic events induced by exposure to the antimetabolite 1-beta-D-arabinofuranosylcytosine (ara-C). Mutant cells produce a truncated M(r) 29,000 protein that interferes with the function of normal c-Jun (and c-Fos) proteins through a quenching mechanism. Parental U937, cells expressing TAM67, and cells carrying only the empty vector (pMM) were exposed to ara-C for 6 h, and apoptosis was monitored by cell morphology as well as qualitative and quantitative assays of DNA damage. No differences in apoptosis could be detected between the three cell lines at any of the ara-C concentrations evaluated. In addition, ara-C concentrations > or = 1.0 x 10(-6) M were equally inhibitory to the clonogenic growth of U937 and TAM67-expressing cells. In contrast, lower concentrations of ara-C (i.e., < 5.0 x 10(-7) M) were significantly less inhibitory to mutant U937 cell colony formation than to their parental counterparts. The reduced sensitivity of TAM67-expressing cells to low concentrations of ara-C could not be attributed to biochemical or cytokinetic factors, since the two cell lines were indistinguishable with respect to 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP) formation, ara-CTP:dCTP ratios, and S-phase fraction. However, a significantly lower percentage of TAM67-expressing cells exposed to submicromolar concentrations of ara-C exhibited features associated with a differentiated monocytoid phenotype (i.e., increased plastic adherence and CD11b expression) compared to their parental counterparts. Lower concentrations of ara-C were also significantly less effective in decreasing the percentage of S-phase cells and in down-regulating c-myc mRNA levels in the mutant line, events associated with induction of leukemic cell differentiation. Finally, ara-C-induced up-regulation of c-jun message and protein was markedly attenuated in TAM67-expressing cells, findings consistent with a c-jun dominant-negative model. Collectively, these findings suggest that dysregulation of c-jun in U937 cells antagonizes low-dose ara-C-mediated cellular maturation but does not prevent higher concentration of this agent from triggering apoptosis. They also raise the possibility that separate aspects of the antiproliferative actions of ara-C may be differentially regulated by c-jun.

The induction of programmed cell death, or apoptosis, involves activation of a signalling system, many elements of which remain unknown. The sphingomyelin pathway, initiated by hydrolysis of the phospholipid sphingomyelin in the cell membrane to generate the second messenger ceramide, is thought to mediate apoptosis in response to tumour-necrosis factor (TNF)-alpha, to Fas ligand and to X-rays. It is not known whether it plays a role in the stimulation of other forms of stress-induced apoptosis. Given that environmental stresses also stimulate a stress-activated protein kinase (SAPK/JNK), the sphingomyelin and SAPK/JNK signalling systems may be coordinated in induction of apoptosis. Here we report that ceramide initiates apoptosis through the SAPK cascade and provide evidence for a signalling mechanism that integrates cytokine- and stress-activated apoptosis.

Tam-67 is an amino-terminal deletion mutant of c-Jun (delta3-122) lacking most of the c-Jun transactivation domain, which has been shown previously to function in a transdominant fashion to inhibit c-Jun-induced transactivation and cellular transformation. In order to create a ligand-dependent dominant negative repressor of AP-1, we have constructed a fusion of the TAM-67 gene with the ligand binding domain of the estrogen receptor. Fusion of TAM-67 with the ligand binding domain of the estrogen receptor produced a 68 kD protein (TAM-67ER) which was immunoprecipitated by c-Jun-specific and estrogen receptor-specific antisera and shown by gel retardation assay to bind oligonucleotides containing an AP-1 sequence. Cotransfection of TAM-67ER and an AP-1-dependent reporter construct into rat embryo cells demonstrated ligand specific inhibition of AP-1 transactivation. In the absence of hormone, TAM-67ER produced complete inhibition of c-Jun-induced AP-I transactivation. This inhibition was relieved by treatment with estradiol but not by treatment with tamoxifen. In addition, TAM-67ER inhibited activated c-Ha-ras- or c-raf-induced transformation of NIH3T3 cells. However, this inhibition of transformation was not relieved by the addition of estrogen. Thus, TAM-67ER inhibits transactivation in a ligand-dependent manner, but inhibits transformation in a ligand-independent manner. The results suggest that the ligand-dependent transactivation domain of the estrogen receptor (TAF-2) can substitute for the c-Jun transactivation domain absent in TAM-67 to stimulate transactivation. However, TAF-2 cannot substitute for the missing c-Jun transactivation domain to induce cellular transformation.

We have studied the expression and activity of the jun and fos families of transcription factors in a panel of human breast cancer cells. Numerous breast-cancer cell lines showed variable levels of expression of jun and fos family-member RNA, activator protein-1 (AP-1) DNA binding, and transcriptional-activating activities during exponential growth. In all of the breast-cancer lines tested, c-jun RNA and AP-1 DNA-binding activity correlated. In addition, in most breast cancer cell lines AP-1 DNA-binding activity also correlates with AP-1-transactivating activity. However, some breast cancer cell lines have high c-jun RNA expression, high AP-1 DNA-binding activity, and low AP-1-transactivating activity. Such results suggest that in these breast cancer cell lines there exist AP-1 complexes that can bind DNA but cannot activate transcription. Multiple peptide growth factors as well as the tumor promoter 12-0-tetradecanoylphorbol-13-acetate induced the expression of jun and fos family-member RNAs and also increased AP-1 DNA-binding activity and functional AP-1-transcriptional activating activity in MCF7 breast cancer cells. However, treatment with estrogen, a steroid growth factor, failed to increase jun and fos RNA expression and induced minimal increases in AP-1 DNA binding and AP-1-induced transcriptional-activating activity in comparison with that seen after peptide hormone treatment. Thus, mitogenic peptide hormones and the tumor promoter 12-0-tetradecanoylphorbol-13-acetate, but not estrogen, strongly activate the AP-1 transcription factor in breast cancer cells. A dominant-negative mutant of c-jun that specifically inhibits AP-1- transactivating activity in rat fibroblasts inhibited AP-1 transactivating activity in breast-cancer cells and blocked the increase in AP-1-mediated transcription induced by serum or specific growth factors. This dominant-negative mutant also inhibited MCF7 colony formation, indicating that expression of this AP-1 inhibitor suppressed the proliferation of these breast cancer cells. Such results suggest that growth factor-induced proliferation of breast cancer cells can possibly be blocked by inhibiting AP-1-transactivating activity.

Although the c-jun oncogene is an integral part of the AP-1 transcriptional complex implicated in the process of tumor promotion, its role in the pathogenesis of human tumors is unknown. We analyzed the expression and function of cJun in 110 non-small cell lung cancer (NSCLC) primary and metastatic tumors, histologically atypical areas from the surrounding lung, and 10 NSCLC cell lines to examine the role of cJun in lung carcinogenesis. cJun was expressed in primary and metastatic lung tumors in 31% of cases, with no association with survival. Whereas normal conducting airway and alveolar epithelial in general did not express cJun by immunohistochemistry, histologically atypical areas were frequently positive for cJun, regardless of the status of the corresponding tumor. Multiple members of the jun and fos gene families were frequently expressed at the mRNA level in vitro, with detectable functional activity (as defined by AP-1-specific DNA binding and/or transactivation of an AP-1-driven reporter construct) present in all 10 NSCLC cell lines examined. Although tumor-promoting phorbol esters had little effect on c-jun expression, serum stimulation generally resulted in significant c-jun induction in NSCLC cell lines. These data show that cJun expression is altered early during human lung carcinogenesis and that cJun may function as a mediator of growth factor signals in NSCLC.

Lung cancer is a major cause of mortality in the United States and accounts for the majority of all cancer deaths in both men and women. It is hoped that through broadening our understanding of the mechanisms involved in transformation of bronchial epithelial cells we will be able to improve methods of diagnosis and treatment of this disease, with the ultimate goal of reducing on lung cancer mortality. A knowledge of the molecular mechanisms involved in processes such as cell division and differentiation is paramount to this task, because it is known that aberrant responses to growth factors or cytokines found in the normal cellular milieu can lead to abnormal cell growth and/or transformation. Signals initiated at the cell membrane by tumor promoters, growth factors, or cytokines are transduced from the cell membrane to the nucleus and are, in part, mediated centrally by transcription factors encoded by nuclear protooncogenes. The transcription factors myc, jun, and fos have been characterized in both normal and transformed lung epithelial cells through detailed studies using cell lines. In this manuscript, we review what is known about the expression and regulation of these nuclear protooncogenes in normal and malignant epithelial cells of the lung, and their role in the development of lung cancer.

We analyzed the expression of the retinoblastoma (Rb) gene in a group of ovarian neoplasms previously characterized for mutations in the p53 suppressor gene and the Ki-ras oncogene. Using immunohistochemical techniques, a total of 59 ovarian neoplasms spanning the histiologic spectrum from benign to malignant were examined for the expression of the Rb protein. All benign cystic adenomas and low malignant potential tumors exhibited normal expression of the Rb protein. Abnormalities in Rb protein staining were noted in 3 of 22 (14%) ovarian carcinomas. The staining patterns included tumors that were totally or focally negative for Rb protein. One tumor focally expressed Rb. This tumor demonstrated a direct juxtaposition of sections of Rb expressing and nonexpressing malignant epithelial cells. Two of the three tumors with abnormal Rb expression also had p53 mutations and staining on serial sections demonstrated that selected ovarian cancer cells possessed mutations in both oncogenes. These data suggest that the loss of Rb gene expression may play a role in the pathogenesis of a small number of invasive ovarian malignancies, but not in noninvasive ovarian neoplasms.

Tumor promoters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF) induce neoplastic transformation, elevated c-jun protein expression, and activator protein-1 (AP-1)-dependent gene expression in JB6 mouse epidermal cells sensitive to tumor promoters (clone 415a P+ cells). In contrast, JB6 cells resistant to tumor promoter-induced transformation (clone 307b P- cells) exhibit a greatly reduced TPA or EGF inducible c-jun expression and AP-1 activity. We have recently shown that induced AP-1 is necessary for tumor promoter-induced transformation of P+ cells because introduction of a dominant negative c-jun mutant into P+ cells inhibits both AP-1 dependent transactivation and the transformation response to tumor promoter. The intent of the investigation presented here was to test the hypothesis that elevation of AP-1 activity is sufficient to cause progression to the P+ phenotype in P- cells or to the transformed phenotype in P+ cells. Clonally derived P+ and P- recipient cells transfected with a human c-jun expression construct and overexpressing c-jun protein were tested for progression by assaying for constitutive or inducible anchorage independent phenotype and nude-mouse tumorigenicity. Overexpression of c-jun did not produce progression in P- cells but did increase the probability of progression in P+ cells (two of five transfectant cell lines progressed to the tumor phenotype). In addition, c-jun overexpression did not increase AP-1 activity in any of the P-/c-jun transfectants or in the two of five P+/c-jun transfectants that acquired the transformed phenotype. The P+/c-jun transfectants that showed elevated AP-1 activity did not progress to the tumor phenotype, demonstrating that an increase in AP-1 activity is insufficient for this progression. Since P(+)-to-tumor phenotype progression occurred in cells overexpressing c-jun but not AP-1, we propose that P(+)-to-transformed phenotype progression is c-jun dependent and AP-1 independent.

In NIH3T3 cells expressing active Raf-1 protein serine/threonine kinase (PSK) c-jun expression is constitutive while c-fos expression is attenuated. This alteration prompted us to determine whether oncogene transformation would render cells differentially sensitive to growth inhibition by a dominant negative mutant of c-jun, TAM 67. Growth inhibition was observed in three types of assays: (1) transfection of TAM 67 into cells stably transformed by a variety of oncogenes, (2) cotransfection of TAM 67 with oncogene expression plasmids into NIH3T3 cells and (3) titration of oncogene-expressing retroviruses on cells stably expressing TAM 67. The results clearly demonstrate that Raf-1 dependent oncogenes, which include receptor protein tyrosine kinases (PTKs)-, intracellular PTKs- and Ras-derived genes share the Raf phenotype of constitutive c-jun expression, attenuated c-fos induction, and high sensitivity to growth suppression by TAM 67. Additionally, the intracellular PSK oncogene, mos and the nuclear oncogenes c-myc, c-fos, and SV40 T antigen were TAM 67-sensitive for transformation. This universal pattern of altered growth regulation in oncogene transformed fibroblast cell lines highlights the potential usefulness of c-jun based inhibitors for control of tumor cell growth.

Gynecologic malignancies, representing 13% of all cancers affecting women, have a major impact on women's health. Cervical, endometrial, and ovarian cancers comprise the majority of these tumors and contribute significant morbidity and mortality to the female population. While cervical and endometrial cancers can be detected early in their development, sadly, many patients present with advanced disease, as do the majority of patients with ovarian cancer. Unfortunately, advanced cases of these malignancies are usually lethal despite modern therapeutic modalities. In order to impact upon these grim statistics, gynecologic researchers have turned to molecular biology in an attempt to elucidate the etiology of these cancers. Recent research describing dominant oncogene and tumor suppressor gene mutations common to these malignancies is providing a basis for the molecular genesis of these cancers. This information should offer new avenues for the development of early detection and chemoprevention, as well as novel treatment strategies.

Previous studies have shown that phorbol ester induced macrophage differentiation in the leukemic cell line U-937 is tightly linked to the expression of the c-jun protooncogene and the generation of AP-1 transcriptional activity. We expressed the c-jun protooncogene in U-937 cells to examine the role of c-jun in the differentiation of these cells. AP-1 DNA binding activity and the expression of AP-1 controlled downstream genes were increased in all clones expressing exogenous cJun. More importantly, these transfectants showed evidence of differentiation as measured by the acquisition of phagocytic capacity, although they did not develop morphological changes associated with differentiation such as adherence. Furthermore, they became committed to terminal differentiation after significantly shorter exposures to phorbol esters than did control cell lines. These data show that cJun expression induces partial differentiation along the macrophage pathway in U-937 cells.

The AP-1 transcriptional activating complex, made up of Jun and Fos protein, is involved in controlling many cellular processes such as cell proliferation, differentiation and transformation. We have previously characterized a dominant-negative mutant of c-Jun called TAM-67 which forms dimers with c-Jun and c-Fos, and binds DNA as a homodimer or heterodimer with c-Jun or c-Fos. This dominant-negative mutant is a potent inhibitor of AP-1 mediated transactivation, as well as c-jun/ras and TPA/ras-induced transformation. The present report describes experiments designed to elucidate the exact molecular mechanism of this dominant-negative inhibitor. The DNA binding kinetics of both TAM-67:TAM-67 homodimers as well as TAM-67:Fos heterodimers were studied and compared to those of c-Jun and other transactivation-deficient mutants of c-Jun. These studies demonstrated that the TAM-67 proteins have similar DNA binding kinetics to c-Jun and other Jun mutant proteins. Thus, the deletion of the amino-terminal end of the Jun protein does not significantly alter the protein's affinity for DNA. In addition, to determine whether TAM-67 functions through the formation of homodimers, or through interactions with endogenous c-Jun or c-Fos, we constructed a pair of chimeric proteins made by replacing the leucine zipper of TAM-67 with the leucine zippers of GCN4 and c-Fos. These chimeric proteins, termed TAM/GCN4 and TAM/Fos, were then tested for their ability to bind DNA, inhibit c-Jun-induced transactivation, and inhibit TPA/ras-mediated transformation. The results of these studies show that while both chimeric proteins bind equally well to DNA, only the TAM/Fos protein, and not the TAM/GCN4 protein, inhibits AP-1-induced transactivation and TPA/ras-induced transformation. When compared to the TAM-67 protein, the TAM/Fos protein is an equally potent inhibitor of transactivation and transformation. These results suggest that TAM-67 inhibits AP-1-mediated processes through a 'quenching' mechanism by inhibiting the function of endogenous Jun and/or Fos proteins. The implications of these mechanistic findings on the development of potent inhibitors of signal transduction pathways are discussed.

We have stably expressed a trans-activation suppressing deletion mutant of the human c-jun gene (TAM-67) in the malignant mouse epidermal cell lines 10Gy5 and PDV. Expression of the p26 mJUN protein blocked both constitutive and inducible transcriptional trans-activation of several AP-1 responsive reporter chloramphenicol acetyltransferase constructs. p26 mJUN was able to block both 12-O-tetradecanoylphorbol-13-acetate (TPA) and okadaic acid induced expression of the mouse stromelysin gene in 10Gy5 cells and TPA induced expression of the urokinase-type plasminogen activator gene in PDV cells as determined by Northern analyses. Both genes contain TPA response elements in their promoter regions and are known to be AP-1 responsive. The presence of p26 mJUN in nuclear extracts, as determined by Western blotting, did not detectably alter the DNA binding activity of endogenous AP-1 as determined by gel shift analysis with an oligonucleotide containing a single high affinity AP-1 binding site. UV cross-linking studies coupled with Western analyses identified DNA bound cJUN but not mJUN in nuclear extracts of stably transfected cell lines, suggesting that the mutant JUN protein may exert some of its antioncogenic effects in malignant mouse epidermal cells by a mechanism(s) not involving DNA binding. Malignant mouse epidermal cells which stably expressed the mutant JUN protein were not only inhibited in their AP-1 trans-activation response, but also in their ability to form s.c. tumors in nude mice. These results indicate that inhibition of AP-1 mediated transcriptional trans-activation alone can be sufficient to suppress the tumorigenic phenotype in a subset of malignant mouse epidermal cells.

In an effort to define the pathogenic relationship between ovarian neoplasms spanning the clinicopathological spectrum from benign to malignant, the incidence of Ki-ras and p53 mutations was determined in 20 ovarian cystadenomas, 20 low malignant potential (LMP) tumors of the ovary, and 23 ovarian carcinomas. Using DNA extracted from paraffin embedded tissue, polymerase chain reaction amplification, designed restriction fragment length polymorphism analysis, and DNA sequencing, 1 cystadenoma (5%), 6 LMP tumors (30%), and 1 ovarian carcinoma (4%) demonstrated an activated Ki-ras gene. All of the Ki-ras mutations identified except one were GGT to GAT transversions at codon 12. One LMP tumor demonstrated a CAA to CAC transversion at codon 61. Using polymerase chain reaction/single strand conformational polymorphism, DNA sequencing, and immunohistochemistry, 11 ovarian carcinomas (48%) demonstrated a p53 mutation. These mutations included 5 missense, 2 nonsense, and 1 frameshift mutation located within exons 6-8 and 3 mutations that were identified only by immunohistochemical staining. No p53 mutations could be identified in cystadenomas or LMP tumors. Clinically, the presence of either a Ki-ras or p53 mutation was associated with advanced stage disease. The pattern of Ki-ras and p53 mutations appears to distinguish LMP tumors from invasive carcinomas and suggests that they may be separate biological entities.

With the overall incidence of lung cancer increasing and little progress being made in effective treatment, new approaches to this disease, such as early detection and prevention, are desperately needed. Rapid advances in molecular oncology over the past decade have provided us with new insights into the pathogenesis of lung cancer. Characterization of molecular changes in growth-regulating genes in lung cancer cells has been a first step toward identification of molecular markers of early lung cancer. In addition, we have made great strides in our understanding of the process of tumor promotion in lung cancer by the identification of specific substances as tumor promoters and the biochemical pathways by which they function. These insights will provide us with a beginning for the development of screening assays, early detection tests, and chemopreventive agents, hopefully allowing us to make an impact on lung cancer mortality.

Jun and Fos proteins are DNA-binding proteins that are involved in the control of gene expression through transcriptional regulation. We have made a deletion mutant of the c-jun gene that lacks amino acids 3-122 of c-jun, and thus is missing the major transactivation domain of c-jun, but retains the DNA-binding and leucine zipper domains. Unlike c-Jun, the mutant protein is unable to stimulate the transcription of an AP-1 responsive gene, and unlike c-jun this mutant gene is unable to transform rat embryo cells in cooperation with an activated ras gene. However, this mutant protein blocks in vitro DNA binding of Jun-Jun homodimers and Jun-Fos heterodimers, transcriptional activation induced by c-jun or c-fos and transformation of rat embryo cells induced by an activated ras gene and a deregulated c-jun or c-fos gene. In addition, transformation of rat embryo cells induced by an activated ras gene in the presence of the tumor promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA) or by ras plus SV40 large T antigen is also inhibited by this dominant-negative mutant, suggesting that a member of the jun or fos family is involved in the pathways leading to transformation in these systems as well. The possible molecular mechanisms by which this dominant-negative mutant of c-jun blocks the functions of wild-type jun and fos family members are discussed.

As we expand our knowledge of the initiators and promoters of lung cancer, early detection and intervention strategies show great potential in individuals at high risk, especially smokers and exsmokers. Documented mutations of dominant oncogenes and tumor suppressor genes in human lung cancer cells may represent important steps in the pathogenesis of invasive cancer. The precise molecular events and their sequence that lead to tumor promotion in lung cancer, however, are less well understood. Chemointervention with agents like the retinoids may halt proliferation of cancer cells prior to the development of metastatic competence. Use of anti-growth-factor therapy and peptide hormone antagonists may also have a role in intervention approaches. This paper reviews present understanding of the initiation and promotion of lung cancer, as well as preventive strategies currently proposed for patients at risk.

Recent studies of the molecular biology of lung cancer have identified multiple abnormalities. Despite this vast cataloging of genetic lesions, the chronology of these events and those which occur early remains essentially unknown. This review summarizes the genetic abnormalities in lung cancer cells, including mutation, amplification, and overexpression of dominant protooncogenes as well as deletion and mutation of recessive oncogenes. In addition, possible candidate genes exist which may participate in the early activation events of lung cancer, and evidence for their role in the early development of cancer is discussed. These lesions may be helpful in developing strategies to screen for lung cancer.

The c-, L-, and N-Myc nuclear phosphoproteins share several highly conserved regions that partially overlap putative functional domains of the c-Myc protein. All three myc oncogenes can cooperate with an activated ras gene to transform primary rat embryo cells (REC), and deregulated expression of c- and L-myc can block differentiation of murine erythroleukemia (MEL) cells. In the present study, we demonstrate that N-myc also can block MEL cell differentiation, and we identify regions within the c-Myc protein that are necessary for inhibition of MEL differentiation. C19 MEL cells were transfected with six human c-myc genes which were partially deleted in different areas of the coding region. Four of the genes lack sequences that overlap either the putative transcriptional activation domain, the helix-loop-helix motif, or the leucine zipper motif and were previously shown to have lost REC cotransforming activity (J. Stone, T. DeLange, G. Ramsay, E. Jakobovitz, J.M. Bishop, H. Varmus, and W. Lee, Mol. Cell. Biol., 7: 1697-1709, 1987). In this study, we demonstrate that they also fail to inhibit N,N'-hexamethylene-bis-acetamide-induced differentiation of MEL cells. In contrast, two partially deleted c-myc genes, one lacking a short NH2-terminal region and the other lacking 118 amino acids at the center of the coding region, which were fully active in REC cotransformation, also exhibited full activity associated with the former and only partial activity with the latter in blocking MEL differentiation. We conclude that the mutated genes tested in this study behave similarly in inhibition of MEL cell differentiation and in REC cotransformation.

The complex process of epithelial carcinogenesis is composed of discrete biologic events including the early activation events of "initiation" and "promotion." For lung cancer, these events are only now being elucidated. Despite the identification of possible target genes and their mutations, the "initiation" events for lung cancer remain poorly understood. The identification of these "initiation" events is a crucial step toward the development of practical molecular markers for early detection of this disease. The reversible process of tumor promotion remains somewhat enigmatic but is a promising target for chemoprevention. A wide range of substances, including asbestos and various substances in cigarette smoke, behave as tumor promoters for lung cancer. They appear to promote tumor formation by inducing cellular proliferation mediated in part by growth factors. The intracellular signals these factors provide are ultimately translated into cellular growth via steps involving nuclear transcription factors. Early response genes such as the jun and fos gene family members encode such nuclear transcription factors which are expressed in lung cancer cells and primary bronchial epithelial cells. The expression of these transcription factors is highly responsive to stimulation by growth factors including serum, transforming growth factor, and gastrin-releasing peptide. A more thorough understanding of this process will allow the development of molecular and/or pharmacologic antagonists that can interfere with the biologic process of tumor promotion and therefore function as chemoprevention agents.

The fact that routinely effective treatments for disseminated lung cancer are not available has prompted the search for effective early detection systems. It is important to identify lung cancer while it is still confined to the bronchial epithelium and is potentially curable with local modalities. We have previously reported on an immunologically based assay to identify antigens expressed on shed bronchial epithelial cells. This assay resulted in a statistically significant correlation of immunostaining with the eventual development of lung cancer 2-4 years prior to routine clinical detection. Attempts to further improve this approach require an understanding of the basis for its success. Based on the work of Hakomori and coworkers, this difucosylated Lewis X structure would be a likely marker of carcinogenic transformation of the bronchial epithelium. In fact, an antibody to this structure was useful for sputum immunocytochemistry analysis for early lung cancer detection. Other carbohydrate structures would also be reasonable markers to evaluate for early detection application, based on the known pattern of expression of these structures in fetal, dysplastic, and neoplastic lung tissue. Another antibody used for sputum immunostaining recognizes a 31-kd protein structure; the antibody is not a known member of a likely class of early detection targets. The reported cases of lung cancer missed by the immunostaining approach included principally adenocarcinoma of the lung, suggesting that the addition of a marker(s) of that type of morphologic differentiation should be considered. Markers to dissect the various forms of lung adenocarcinoma are being characterized and are available for evaluation in early detection applications.(ABSTRACT TRUNCATED AT 250 WORDS)

The need for validated intermediate end point markers to facilitate lung cancer chemointervention research is compelling. Three major classes of lung markers are relevant for this application. Since lung cancer includes four distinct histologies, markers that map degrees of histologic differentiation are important. Many of the markers for squamous differentiation overlap with the candidates for application in the study of head and neck cancer. Production of tissue-specific cell products especially for surfactant or CEA is of interest, because the gene structure is known and many differentiation-related polymorphisms exist. This strategy would be useful for adenomatous type tissue. A second type of marker is the broad group of differentiation markers. The carbohydrate or blood group-like antigens comprise a representative example. Carbohydrate structures are expressed in a specific sequence during fetal processes, and this sequence appears to reverse with the development of a cancer. Retrodifferentiation of specific differentiation markers is the basis of a major effort to effect earlier lung cancer detection using sputum immunocytochemistry. The final class includes markers which affect either positive or negative aspects of growth. Candidates in this area include growth factors or their receptors, or genes that regulate growth. If the intermediate end point marker reflects tumor biology and that biology is in the causal path of tumor progression, serial observation of that parameter should indicate the success of the intervention. In all three of these examples, the clinical material to be analyzed could be sputum specimens, bronchial biopsies or resected lung tissue. Systematic analysis of these markers in context of intervention trials is required to validate their utility.(ABSTRACT TRUNCATED AT 250 WORDS)

Terminal differentiation of the leukemic cell lines U-937 and HL-60 by 12-O-tetradecanoylphorbol-13-acetate is accompanied by marked changes in gene expression. In this study, we demonstrate that the expression of jun and fos gene family members is induced with variable kinetics during 12-O-tetradecanoylphorbol-13-acetate induced differentiation, with c-jun expression best paralleling differentiation. The generation of AP-1 complexes, as measured by DNA binding activity, closely parallels morphological differentiation. Furthermore, the ability of these complexes to regulate gene expression is demonstrated by increased transcription from an AP-1 driven reporter construct and marked increases in the expression of endogenous AP-1 regulated genes. Differentiation assays using water soluble phorbol esters reveal that differentiation becomes irreversible soon after AP-1 appears. This tight correlation between c-jun expression, the generation of AP-1 activity, and differentiation suggests a critical role for this gene and transcriptional complex during this process.

We explored the state of the p53 gene in gastric cancer. Using one or more methods, we examined 15 specimens from primary carcinomas (14 tumors, one cell line), five cell lines derived from metastases, and seven paired samples of nonmalignant gastric mucosa. Sequence analyses of complementary DNA containing the entire p53 gene open reading frame demonstrated abnormalities in one of five samples from primary tumors and in all five samples from metastases. The single cell line derived from a primary carcinoma had no abnormality of the gene. The six abnormalities included four point mutations, one base-pair deletion resulting in a frame shift, and a 24 base-pair deletion caused by an intronic point mutation (as determined by sequence analysis of genomic DNA). Four of the six mutations mapped to regions highly conserved among species or involved in simian virus 40 T-antigen binding. Restriction fragment length polymorphism studies confirmed that chromosome 17p allelic deletions occur only in a minority of primary tumors, but that they may occur more frequently in metastases. Northern blotting and ribonuclease protection assays detected only a fraction of the p53 gene abnormalities detected by sequencing. Our findings indicate that mutations of the p53 gene are relatively rare in primary gastric tumors but appear to be relatively frequent in cell lines derived from metastatic lesions. Our results may help in understanding the molecular events associated with progression and metastasis in gastric carcinoma.

Molecular analysis of the human proto-oncogene L-myc revealed a complex pattern of gene expression including alternative splicing and polyadenylation site selection of mRNA, giving rise to at least four mRNAs. These mRNAs in turn can code for several proteins. In this report, we characterize and define the origins of the major L-myc proteins. In vitro translation revealed that (i) two L-myc proteins (p59 and p65) were derived through alternative translational initiation at a non-AUG (CUG) site in intron 1 and at an AUG site in exon 2 of L-myc, and that (ii) extensive post-translational phosphorylation of these proteins yielded three additional proteins (p60, p66, and p68). Transfection experiments in rat embryo cells revealed the in vivo existence of this unusual CUG-initiated protein and demonstrated that it possessed transforming activity. Further, immuno-precipitation using high titered anti-L-myc peptide antisera, of two L-myc expressing small-cell lung cancer cell lines revealed three major L-myc proteins (p60, p66 and p68) all of which were derived from extensive phosphorylation of a p59 protein.

Hyponatremia in patients with small cell lung cancer can be caused by tumor production of arginine vasopressin (AVP) and result in the syndrome of inappropriate antidiuretic hormone. In evaluating the expression of AVP mRNA from tumor and tumor cell line specimens from five patients with small cell lung cancer and hyponatremia (presumed to have the syndrome of inappropriate antidiuretic hormone), we found that the tumors and tumor cell lines from two of these five patients expressed AVP mRNA. The RNA samples from the three patients with undetectable AVP mRNA expressed abundant atrial natriuretic factor (ANF) mRNA. Analysis of specimens from three patients with small cell lung cancer and normal serum sodium levels revealed no detectable AVP mRNA expression, and samples from only one of these three patients' specimens expressed detectable ANF mRNA. The AVP and ANF peptide levels in lysate preparations of the tumor cell lines from four of these patients were tested by radioimmunoassay and confirmed the gene expression data. These studies demonstrate ectopic production of ANF mRNA in small cell lung cancer specimens from patients with this cancer and the syndrome of inappropriate antidiuretic hormone. These findings will be of particular interest if future studies demonstrate that ectopic ANF production can cause sodium abnormalities in patients with small cell lung cancer.

Allele loss is a hallmark of chromosome regions harboring recessive oncogenes. Lung cancer frequently demonstrates loss of heterozygosity on 17p. Recent evidence suggests that the p53 gene located on 17p13 has many features of such an antioncogene. The p53 gene was frequently mutated or inactivated in all types of human lung cancer. The genetic abnormalities of p53 include gross changes such as homozygous deletions and abnormally sized messenger RNAs along with a variety of point or small mutations, which map to the p53 open reading frame and change amino acid sequence in a region highly conserved between mouse and man. In addition, very low or absent expression of p53 messenger RNA in lung cancer cell lines compared to normal lung was seen. These findings, coupled with the previous demonstration of 17p allele loss in lung cancer, strongly implicate p53 as an anti-oncogene whose disruption is involved in the pathogenesis of human lung cancer.