Carbon quantum dots/block copolymer ensembles for metal-ion sensing and bioimaging.

Fluorescent carbon quantum dots (CQDs) rich in amine moieties that can be easily protonated under acidic conditions were electrostatically interacted with diblock copolymer poly[sodium(carboxylate sulfamate) isoprene]-b-poly(ethylene oxide) (CSS-IEO-3) possessing negatively charged sulfamate and carboxylate species on the CSS part of the diblock copolymer. The so-formed CQDs/CSS-IEO-3 material was found to be stable in a neutral and high salinity environment, which is critical when aiming for biological applications. Moreover, the photoluminescence of CQDs within CQDs/CSS-IEO-3, with emission at 420 nm upon excitation at 320 nm, was unaffected by pH and salinity changes, denoting the absence of significant aggregation phenomena and highlighting the potential of CQDs/CSS-IEO-3 for bioimaging. The photoluminescence of CQDs/CSS-IEO-3 was employed to develop a selective and sensitive method for the facile detection of Fe3+, in the presence of other metal salts, with a detection limit of 8.37 µM. Furthermore, integration of bovine serum albumin (BSA) furnished the three-component CQDs/CSS-IEO-3·BSA nanoensemble, without causing cytotoxicity when subjected to RKO human colon adenocarcinoma cell line. Evidently, confocal fluorescence microscopy allowed the imaging of CQDs/CSS-IEO-3·BSA within living cells, and illustrated its application potential for bioimaging and drug delivery.

High levels of phosphorylated c-Jun, Fra-1, Fra-2 and ATF-2 proteins correlate with malignant phenotypes in the multistage mouse skin carcinogenesis model.

Analysis of the functions of AP-1 transcription factor in cellular systems has shown its key role as a mediator of oncogenic signals. The employment of suitable animal model systems greatly facilitates the study of changes in the composition and activity of the AP-1 complex. Here, we have analysed the quantitative and qualitative changes of AP-1 at different stages of carcinogenesis in mouse skin cell lines, derived from tumours induced by chemical mutagens. The findings of this study suggest that elevated AP-1 DNA binding and transactivation activity characterize the carcinoma cell lines, most notably the highly malignant spindle carcinomas. In addition, increased amounts and post-translational modifications of c-Jun, Fra-1, Fra-2 and ATF-2 proteins account for a high percentage of the increased AP-1 activity. Remarkably, high levels of phosphorylated ATF-2 protein were detected in malignant cell lines, indicating a novel role of ATF-2 in tumour progression. c-Jun and ATF-2 proteins are phosphorylated by highly active JNK kinases present in tumour cells. Finally, our results indicate distinct functions for different AP-1 components in the promotion and progression of mouse skin tumours. Oncogene (2000) 19, 4011 - 4021.

Mutation of a phosphorylation site in the DNA-binding domain is required for redox-independent transactivation of AP1-dependent genes by v-Jun.

The ability of the nuclear oncoprotein Jun to activate transcription is controlled both by level of DNA binding and by the activity of its transactivation domain. Control of DNA binding is achieved by two mechanisms: phosphorylation and redox regulation. Mutation of Ser-226 inhibits phosphorylation of the DNA binding, resulting in enhanced DNA-binding and transactivation activity of Jun. In contrast, mutation of Cys-252, which is the target for repression of DNA-binding activity under oxidative conditions, results in a strong decrease of Jun-specific activation of transcription. However, transactivation by c-Jun-Cys-252 is fully restored upon mutation of Ser-226. Both mutations are also found in the oncogenic counterpart of c-Jun, v-Jun, and are the only differences between these proteins in the DNA-binding domain, suggesting that v-Jun escapes down-modulation of DNA binding by both mechanisms. However, inhibition of phosphorylation of Ser-226 is absolutely required for the ability of v-Jun to activate transcription of AP-1-dependent genes in a redox-independent manner.

Regulation of AP-1/DNA complex formation in vitro.

The level of AP-1 DNA-binding activity exhibited in vitro by unfractionated extracts of Hela nuclei can be stimulated by a low molecular weight fraction from rabbit reticulocyte lysate. Stimulation also requires a heat labile component of the nuclear extract, probably a protein. Stimulated and unstimulated extracts with high and low AP-1 DNA-binding activities contain the same levels of proteins reactive with antisera against Jun and Fos, proteins which are shown to be involved in the AP-1/DNA complexes detected in vitro. The low molecular weight fraction from reticulocyte lysate can be substituted by the reducing agent dithiothreitol (DTT) in the stimulation reaction and conversely oxidised glutathione greatly reduces formation of AP-1/DNA complexes. The binding activities of transcription factors SP-1, NF-1 and CBP are unaffected by DTT or oxidised glutathione. These observations, taken together, suggest that the efficiency with which pre-existing Fos and Jun proteins can bind an AP-1 target sequence in vitro can be controlled by a nuclear activity which is sensitive to oxidation/reduction and that this control mechanism is specific for AP-1.

Differential potency and trans-activation of normal and mutant T24 human H-ras1 gene promoters.

We have employed a short-term transfection assay system in which we monitored the transient expression of the chloramphenicol acetyltransferase (CAT) gene linked to the promoter region of the normal and mutant T24 H-ras1 gene or the human epsilon-globin gene in Chinese hamster lung (CHL) cells or cells derived from them which carry and express one or the other of the polyoma virus early genes. Our findings can be summarized as follows: (i) The mutant T24 H-ras1 promoter region behaves as a stronger promoter than the H-ras1 gene in all these types of cells as well as in rat 208F fibroblast cells. (ii) In CHL cells expressing the polyoma large T antigen the normal and mutant T24 Ha-ras1 promoters are not trans-activated in these cells and only a 2.5-fold activation of the epsilon-globin promoter is observed. (iii) In cells expressing the polyoma middle T antigen both the normal and mutant H-ras1 are trans-activated whereas transcription from the epsilon-globin promoter is not affected when compared to the normal CHL cells. (iv) In cells expressing the polyoma small T antigen the normal and mutant H-ras1 as well as the epsilon-globin promoters are trans-activated. We suggest from these data that a tissue-specific element exists in the promoter region of the H-ras1 gene and that the polyoma middle and small T antigens trigger the expression of proteins that trans-activate these promoters.

Phorbol ester-responsive H-ras1 gene promoter contains multiple TPA-inducible/AP-1-binding consensus sequence elements.

We have constructed recombinant DNA plasmids which carry both the aminoglycoside phosphotransferase (aph) gene and the chloramphenicol acetyl-transferase (CAT) gene linked to the human normal or mutant T24 H-ras1 promoter. We have transfected these plasmids into rat 208F fibroblasts using the calcium phosphate technique and selected for stable transformants by geneticin resistance. These transformants expressed CAT activity at low levels. However, when treated with the phorbol ester TPA, CAT levels increased substantially. Cells transfected with recombinant plasmids carrying a promoterless CAT gene did not respond to TPA. We have noted four motifs in the H-ras1 promoter region which resemble TPA-inducible and AP-1-binding consensus sequences. We suggest that AP-1-like proteins may play a role in control of H-ras1 transcription.

Immunohistochemical study of the ras oncogene expression in human bladder endoscopy specimens.

We have used the monoclonal antibody Y13 259 to the ras oncogene p21 protein product in 42 endoscopy specimens from the bladder of 37 patients, in order to determine the ras oncogene expression in different conditions of the urothelium. The examined material included: 27 normal and hyperplastic or dysplastic mucosae and 13 papillomas and transitional cell carcinomas, graded according to Mostofi's classification. Our results showed the following: the normal urothelium sections tended to be negative, while the umbrella cells from the superficial layer always expressed a higher degree of positivity. The majority of the hyperplastic lesions and the papillomas were weakly positive or negative. In contrast, all the dysplastic lesions and the carcinomas of different grades were strongly positive. Our results suggest that elevated expression of ras oncogene may serve as an early marker in the pathogenesis of bladder lesions.

Sp1 specific binding sites within the human H-ras promoter: potential role of the 6 bp deletion sequence in the T24 H-ras1 gene.

Transcriptionally active domains have been identified and located within the 5'-region of the human normal and mutant T24 H-ras1 promoters, and have been characterised by linkage to the coding sequences of the bacterial chloramphenicol acetyltransferase (CAT) gene or by using DNaseI foot-printing analysis of the promoter sequence. It has been shown, using the latter method, that Sp-1 transcription factor binds to six GC sequences within the H-ras promoter. In the present study we have used unfractionated nuclear protein preparations from HeLa cells and a gel retardation assay to analyse specific binding of nuclear protein preparations from HeLa cells and a gel retardation assay to analyse specific binding of nuclear factors to several oligonucleotide sequences of the human H-ras1 promoter. Our data demonstrate the presence of three Spl specific binding sequences in the T24 promoter, one of them containing a Sp-1 consensus GGCGGC absent in the normal H-rasl promoter.

Human immunodeficiency virus long terminal repeat responds to transformation by the mutant T24 H-ras1 oncogene and it contains multiple AP-1 binding TPA-inducible consensus sequence elements.

We have employed a short term transfection assay to examine the response of HIV-1 LTR to transformation by the human normal and mutant T24 H-ras1 genes. The plasmid pBC12HIVCAT which carries the HIV-1 LTR sequences linked to the reporter gene chloramphenicol acetyl transferase (CAT) was transfected into rat 208F fibroblasts and their derivatives RFHO6N1-1 and RFHO6T1-1 transfectants. RFHO6N1-1 and RFHO6T1-1 express an exogenous human normal or mutant T24 H-ras1 gene respectively. Expression of the mutant T24 but not the normal H-ras1 gene resulted in increased levels of HIV-1 LTR driven CAT activity. We have noted four motifs in the HIV-1 LTR region which resemble TPA-inducible and AP-1 binding consensus sequences. Since H-ras1 fos and jun/AP-1 respond to TPA and T24 H-ras1 is known to induce both fos and jun/AP-1 nuclear transcriptional factors, it is possible that the latter genes play a role in HIV-1 transcription. Moreover, H-ras1 oncogene activation may play an important role in HIV gene expression and in the activation of latent HIV.

Expression of ras and myc oncogenes in human hepatocellular carcinoma and non-neoplastic liver tissues.

An immunohistochemical assay was used to assess expression of ras p21 and myc p62 oncogene products in human hepatocellular carcinoma (HCC) and non-neoplastic liver tissues. The monoclonal antibodies Y13 259 and Myc1-9E10, specific for ras p21 and myc p62 oncoproteins, were employed on paraffin-embedded sections. Most HCCs showed enhanced ras p21 and myc p62 expression, as indicated by staining intensity. Cirrhotic livers revealed increased myc p62 and occasionally increased ras p21 expression. HBsAg+ hepatocytes showed intense immunostaining for ras p21. Fibrotic, cholestatic, fetal and normal adult liver did not present enhancement of oncoprotein production. We suggest that combined over-expression of ras and myc oncoproteins may be important for the malignant phenotypic alteration in human HCC.

Elevated expression of the myc gene in human benign and malignant breast lesions compared to normal tissue.

Expression of the c-myc gene in human breast lesions and in adjacent normal tissue was studied by immunohistochemical analysis. The previously described monoclonal antibody Myc1-9E10 (1) which recognizes the p62 c-myc protein was used in paraffin tissue sections. A total of 101 cases of breast disease examined included 38 simple and complex cystic disease, 18 simple and hyperplastic fibroadenomas, 36 ductal and lobular carcinomas and 9 in situ carcinomas. Whereas the adjacent normal tissue was slightly positive, 25 out of 38 cystic disease, 7 out of 18 fibroadenoma, 36 out of 36 carcinoma and 9 out of 9 in situ carcinoma specimens showed moderate to high levels of p62 c-myc expression as indicated by staining intensity. These results suggest that the c-myc protein may play a role in breast neoplasia.

Elevated expression of AP-1 activity in human breast tumors as compared to normal adjacent tissue.

The levels of AP-1 activity in human breast lesions and in adjacent normal tissue were studied by a gel retardation assay. A thirty nucleotide long consensus oligonucleotide to the adenovirus E3 gene AP-1 sequence (E3AP-1) was end labelled and reacted with nuclear extracts from breast lesions and adjacent normal tissue. A total of 20 tissue extracts (8 pairs of tumor and normal tissue from the same patient and 4 tumors) were examined. All 12 tumor tissues showed elevated levels of AP-1 as compared to the 8 normal tissues. These results suggest that the AP-1 transcription factor may play a role in breast neoplasia.

Prognostic significance of matrix metalloproteinases 2 and 9 in bladder cancer.

BACKGROUND: The levels of matrix metalloproteinases MMP-2 and MMP-9 (type IV collagenases), which degrade the extracellular matrix of the basement membrane, were evaluated as prognostic indicators of metastasis in urothelial carcinoma. MATERIALS AND METHODS: Quantitative gel zymography and immunohistochemistry were used and compared with clinical data at the follow-up period of 36 months. RESULTS: Zymographical analysis of the levels of MMP-9 and active MMP-2 showed a statistically significant increase with tumor grade and invasiveness. This correlation was confirmed by immunohistochemical analysis of MMP-9 expression. However, the correlation between the levels of both gelatinases with recurrence in superficial tumours or progression in invasive tumours was not statistically significant. CONCLUSION: MMPs may have an important role in the invasion mechanism of urothelial cancer and could be useful prognostic markers for patients with bladder carcinoma. The relationship between MMP-2 and MMP-9 expression and the metastatic potential of bladder carcinoma needs further evaluation in subsequent clinical studies.

Immunohistochemical study of ras oncogene expression in endometrial and cervical human lesions.

The Y13 259 monoclonal antibody to the ras p21 protein was used in an immunohistochemical assay to study the levels of ras p21 in human uterine lesions as compared to normal tissue. Out of 73 hysterectomies obtained we have examined ras p21 expression in separately made sections from the endometrium, the cervix and leiomyomas found in the same specimens. A total of 155 tissue sections were finally evaluated and included: 55 endometrial mucosae (normal, hyperplastic and atrophic), 13 leiomyomas, 60 cervicitis (mild, moderate and severe with or without dysplasia), 3 in situ and 7 invasive carcinoma of the cervix, 12 invasive adenocarcinoma of the endometrium and 5 endometrial adenocarcinomas, which involved the cervical canal. Out of the 73 hysterectomy specimens 27 lesions were malignant and showed elevated expression of ras p21. The remaining 128 were normal or atrophic mucosae and benign or premalignant lesions, which were mostly negative. However, all cases of severe cervicitis and dysplasias and 6 out of the 12 hyperplastic endometrial lesions were found to be moderately or highly positive. Our results suggest that elevated expression of ras oncogenes may play an important role in the development of human uterine lesions.

A molecular signature for oncogenic BRAF in human colon cancer cells is revealed by microarray analysis.

Sporadic colorectal cancer develops through a number of functional mutations. Key events are mutually exclusive mutations in BRAF or RAS oncogenes. Signatures for BRAF oncogene have been revealed in melanoma. In a previous study we have reported a molecular signature for HRAS and KRAS mutations in colorectal cell lines that also showed an EMT phenotype for HRAS. In this study we report a molecular profile for a BRAF oncogenic mutation BRAFV600E in colon using the Illumina 45,000 gene microarray. Key differentially expressed genes have been identified from the array analysis further verified by qPCR analysis. Ingenuity pathway analysis such as microsatellite instability, kinase signalling, apoptosis, WNT and Integrin signalling is presented. MutBRAF transforms cells through cross talk with developmental pathways Hedgehog and Wnt, as well as by deregulation of colorectal cancer related kinase pathways, like PI3K. Differential gene expression of BRAFV600E in colon as compared to those associated with RAS oncogenes is presented, as well as similarities and differences between oncogenic BRAF signatures in colon as compared to thyroid and melanoma are highlighted. Novel selected genes/pathways are validated in cell lines and clinical samples bearing BRAFV600E and may serve as markers/targets for personalised diagnosis/therapy/resistance of colorectal cancer.

Newly established tumourigenic primary human colon cancer cell lines are sensitive to TRAIL-induced apoptosis in vitro and in vivo.

Most data on the therapeutic potential of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) as well as resistance to FAS ligand (FASL) in colorectal cancer have come from in vitro studies using cell lines. To gain a clearer understanding about the susceptibility of patient tumours to TRAIL and FASL, we derived primary human cancer epithelial cells from colon cancer patients. Characterisation of primary cultures PAP60 and MIH55 determined their highly proliferating advantage, transforming capability and tumorigenicity in vitro and in vivo. Although FASL treatment appeared to cause little apoptosis only in the PAP60 primary culture, increased apoptosis independent of p53 was observed in both primary PAP60 and MIH55 and control cell lines Caco-2, HT29 and DLD-1 after treatment with SuperKiller TRAIL. Expression analysis of death receptors (DR) in the original parental tumours, the primary cultures before and after engraftment as well as the mouse xenografts, revealed a significant upregulation of both DR4 and DR5, which correlated to differences in sensitivity of the cells to TRAIL-induced apoptosis. Treating patient tumour xenograft/SCID mouse models with Killer TRAIL in vivo suppressed tumour growth. This is the first demonstration of TRAIL-induced apoptosis in characterised tumorigenic primary human cultures (in vitro) and antitumour activity in xenograft models (in vivo).

ras p21 oncoprotein is autoregulated and acts as a potential mediator of insulin action or the H-ras1 promoter.

Rat fibroblast cells carrying an exogenous normal or mutant T24 human H-ras1 gene were transfected with plasmids carrying the normal or mutant T24 H-ras1 gene promoter linked to the reporter chloramphenicol acetyl transferase (CAT) gene and the cells were treated with insulin. We found that the H-ras1 gene was positively autoregulated and that insulin potentiated the response of the T24 ras p21 to the H-ras1 gene promoter. We have also examined the effect of insulin directly on the H-ras1 promoter by treating stable transfectants obtained after transfection of rat fibroblasts with plasmids carrying the normal or mutant T24 H-ras1 gene promoter linked to the reporter CAT gene and the selectable marker aminoglycoside phosphotransferase (aph) gene. We found that insulin appeared to have no direct effect on the H-ras1 promoter in this case, suggesting that the effect is mediated through the ras p21 oncogene product. We suggest that the mutant T24 H-ras p21 protein mediates the action of insulin.

Role of matrix metalloproteinase-9 in progression of mouse skin carcinogenesis.

Invasion of malignant tumor cells is required for the formation of metastatic colonies. Uncontrolled expression of matrix metalloproteinase (MMP)-2 and MMP-9 is a critical part of the invasive potential of tumor cells and is affected by the balance between the enzymes and the inhibitors secreted by the cell. Here we analyzed the expression and activity of the two gelatinases (MMP-2 and MMP-9) as well as the expression levels of the tissue inhibitor of metalloproteinase (TIMP2)-, in different stages of carcinogenesis using mouse skin cell lines derived from tumors induced by chemical mutagens. Our results suggested that the expression of MMP-9 was implicated in the progression to spindle cell carcinomas in mouse keratinocytes. MMP-2 levels remained steady in all cell lines, whereas levels of TIMP-2 were increased in normal and spindle cells. The AP-1 DNA binding and transcriptional activity on the MMP-9 promoter were increased in the malignant cell lines, indicating the requirement of this binding site for its activation. The results of this study clearly suggested the important role of MMP-9, but not of MMP-2, in the metastatic properties of mouse keratinocytes.

Transformation by the fos or jun oncogene does not increase AP-1 DNA-binding activity.

The c-fos and c-jun proto-oncogenes encode components of the transcription factor AP-1. To determine whether transformation by the v-fos or v-jun oncogene results in alterations in the level or regulation of this factor, we have characterized AP-1 DNA-binding activity in nuclear extracts prepared from v-fos- and c-fos-transformed rat fibroblast cell lines and v-jun-transformed chicken embryo fibroblasts under various growth conditions. During proliferation, the level of AP-1 DNA-binding activity does not differ among the v-fos, c-fos, or v-jun-transformed cells and their normal progenitors, despite constitutive overexpression of the corresponding oncoproteins. Therefore, although necessary, it is not likely that an increase in DNA binding is sufficient for fos or jun transformation. Normal rat and chicken fibroblasts demonstrate very low levels of AP-1 DNA-binding activity when quiescent, and upon serum stimulation a biphasic increase is observed. A similar cyclical pattern is seen in v-fos-transformed cells, but in v-jun-transformed cells AP-1 DNA-binding activity does not fluctuate in response to serum stimulation, which suggests that this level of control may be exerted through the Jun component of the AP-1 complex.

Net, a new ets transcription factor that is activated by Ras.

Ras signaling appears to be mediated in part by transcription factors that belong to the ets gene family. To identify downstream targets for the Ras signal transduction pathway, we have used Ras-transformed mouse fibroblasts to isolate a new member of the ets gene family, net. Net has sequence similarity in three regions with the ets factors Elk1 and SAP1, which have been implicated in the serum response of the fos promoter. Net shares various properties with these proteins, including the ability to bind to ets DNA motifs through the Ets domain of the protein and form ternary complexes with the serum response factor SRF on the fos serum response element, SRE. However, Net differs from Elk1 and SAP1 in a number of ways. The pattern of net RNA expression in adult mouse tissues is different. Net has negative effects on transcription in a number of assays, unlike Elk1. Strikingly, Ras, Src, and Mos expression switch Net activity to positive. The study of Net should help in understanding the interplay between Net and other members of the Elk subfamily and their contribution to signal transduction through Ras to the nucleus.