Biomedical subjects

Z Ronai

Publications and source records attributed to Z Ronai.

At least 109 records · Page 6Linked to original sources

PubMed1995

Sampling method as a key factor in identifying K-ras oncogene mutations in preneoplastic colorectal lesions.

The development of sensitive polymerase chain reaction (PCR)-based techniques in recent years has enabled us to verify the possible presence of mutated oncogenes and tumor suppressor genes in stages preceding tumor formation. Early detection of mutants serves as a powerful tool for detecting exposure to carcinogens and can assist in diagnosis. In studies performed in the past few years, we have identified mutant ras alleles in preneoplastic samples of patients with and without colorectal cancer. Our studies have shown (i) that mutant ras alleles are present at low incidence in normal appearing tissues of patients with colorectal cancer. Such mutations are also found in colonic effluents of patients at risk for developing this tumor type; and (ii) that the method of sampling is critical to ensure true representation of the entire colonic mucosa. The implications of identifying ras mutations in patients without evidence for neoplasma are discussed.

Biomarkers, Tumor↗

PubMed1994

Detection of K-ras mutation in colonic effluent samples from patients without evidence of colorectal carcinoma.

BACKGROUND: K-ras gene mutation appears in more than 50% of patients with colon tumors. Both its frequency and early appearance may qualify this mutation as a potential biomarker. To enable early detection of mutant K-ras alleles, we had previously developed a sensitive polymerase chain reaction (PCR)-based assay, i.e., enriched PCR, which enables detection of one mutant K-ras allele present within 10,000 normal alleles. Using the enriched PCR, we were able to detect mutant K-ras alleles in "normal-appearing" colonic mucosa in patients with colorectal cancer. PURPOSE: A study was initiated to determine whether mutant K-ras alleles could be identified in colonic effluent samples of patients who may be at risk to develop colorectal cancer. METHODS: Over 9 years, colonic effluent samples were collected prior to routine colonoscopy from 39 patients who were apparently free of colorectal cancer. These samples were collected from patients with a family history of colorectal cancer (n = 7), adenomatous polyps (n = 7), previously resected colorectal cancer (n = 5), inflammatory bowel disorders (n = 13), normal colonoscopic examination (n = 6), and familial adenomatous polyposis (n = 1). All of the samples were double coded and analyzed for K-ras gene mutation. RESULTS: Of the 39 patients, seven were found to harbor mutant K-ras codon 12 alleles. Mutations were found in patients with a family history of colorectal cancer (three of seven), adenomatous polyps (one of seven), previously resected colorectal carcinoma (two of five), and familial adenomatous polyposis (one of one). In one case, effluent was found to harbor a mutant K-ras allele 4 years before the patient was diagnosed with colorectal cancer. CONCLUSIONS: (a) Effluent samples contain enough DNA to be detected with enriched PCR. Such samples may well be representative of the entire colon in general as opposed to a localized area such as that usually analyzed during colonoscopy. (b) K-ras gene mutation can be identified in routinely obtained colonic washings of patients who are at risk of developing colorectal cancer. Such mutations were absent in patients with inflammatory bowel disorders and in those who had undergone normal colonoscopic examinations. Detection of K-ras mutation in colonic washings may assist in identifying patients who may be at high risk for developing adenocarcinoma of the colon. IMPLICATIONS: The ability to examine colonic effluents provides a powerful and convenient source of sampling and may be adapted for future large-scale screening.

Adult↗

PubMed1994

UV-responsive element (TGACAACA) from rat fibroblasts to human melanomas.

When taken together, several lines of evidence suggest that the URE-bound proteins are associated with DNA replication. (1) A trans-acting factor of 60 kDa (which may include the 68-kDa URE-binding proteins) was found to be induced by UV and to mediate polyoma DNA replication; (2) the URE was able to compete for the binding of factors that promote polyoma replication in rodent cells; (3) URE-bound proteins are expressed to a higher extent at the S phase of the cell cycle; and (4) they are induced following treatment with aphidicholin. These observations may suggest that the URE may play a role in growth "release" (as opposed to growth "arrest") which would assist in restoring normal growth following DNA damage. It is clear that URE-bound proteins consist of multiple transcription factors, some of which are well characterized (i.e., jun, CREB, and fos families); however, it is likely that the growth release phenomenon we relate to is also mediated by (1) other members of these transcription factor families which have not been identified as yet and (2) a specific combination of known transcription factors which bind to this response element under certain circumstances. This hypothesis is outlined in the enclosed model (Fig. 3).

Animals↗

PubMed1994

Expression and transcriptional activity of AP-1, CRE, and URE binding proteins in B16 mouse melanoma subclones.

The expression and DNA binding activity of members of the activating protein-1 (AP-1) and activating transcription factor (ATF) families of transcription factors were analyzed in sham and ultraviolet (UV)-irradiated subclones of the B16 mouse melanoma cell system. The four subclones we used represent sequential stages in the development and progression of malignant melanoma and exhibit differences in growth and metastatic potential. Western blot analysis revealed differential expression of some AP-1 (c-jun, jun-B, and jun-D) and ATF (43- and 47-kDa cyclic AMP-responsive element binding protein (CREB) family members) in the different subclones; while c-jun expression was noted in the subclones with the greater malignant potential, jun-D was expressed in those with the lesser malignant potential. Furthermore, a delicate balance between the two forms of CREB was noted; the 47-kDa CREB appeared, when expressed exclusively, in subclones that exhibit a greater malignant potential. Electrophoretic mobility shift assays using AP-1, CRE, and UV-responsive element (URE) consensus sequences indicated that distinct complexes were formed with extracts from each of the four subclones. The complexes were competitively inhibited by each of the target sequences used, suggesting that "cross-talk" occurs between some AP-1 and ATF family members in this cell system. Moreover, a multimer of the URE sequence, cloned upstream of a chloramphenicol acetyltransferase reporter gene, was transcriptionally active and responsive to UV irradiation in two of the four subclones. UV-related transcriptional activation was directly correlated with the expression of a 43-kDa CREB. Together, these observations identify members of AP-1 and CREB families whose expression and activities correlate with the malignant potential of subclones that represent different stages in melanoma development and progression.

Animals↗

PubMed1994

PEBP2--a modulator of polyoma DNA replication.

Previously, we have shown that integrated copies of polyoma DNA can be induced to replicate in rat fibroblasts (H3 cells) exposed to a DNA-damaging agent. In the current study, we demonstrate that UV-irradiation of mouse fibroblasts (WOP cells), transiently transfected with polyoma DNA, results in repression of polyoma replication. Cotransfection of oligomers representing wild-type but not mutated forms of the PEBP2 target sequence restored levels of viral replication indicating a role of PEBP2 binding proteins in mediating this effect. DNA-binding assays revealed that a different subset of complexes was formed with the PEBP2 target sequence when nuclear proteins from sham and UV-irradiated WOP and H3 cells were compared, suggesting that the activities of PEBP2 binding proteins are differentially regulated upon UV-irradiation in these two cell types. The ability of PEBP2 to modulate polyoma replication following UV-irradiation in WOP cells suggests a potential role of PEBP2 proteins in the cellular response to DNA damage.

Animals↗

PubMed1994

Regulation of c-jun by lung carcinogens in Clara cells of hamsters.

In vitro differentiated hamster Clara cells were used to study the effects of lung carcinogens on the regulation of the c-jun oncogene. Northern blot analysis revealed a decrease in the expression of jun transcripts 24 h following the exposure of Clara cells to the direct acting forms of benzo[a]pyrene (BPDE*) or 5-methylchrysene (5MeCDE). To determine whether this decrease was mediated at the transcriptional level, we have used CAT reporter constructs driven by nested deletions of the 5' non-coding regulatory region of the c-jun oncogene. While BPDE was capable of activating certain regulatory domains of the c-jun promoter, this activation was not observed with either 5MeCDE or the less active lung carcinogens BADE or 6MeCDE. Analysis of enhancer elements identified the SP1 target site as a strong silencer after BPDE treatment. While positive regulatory element(s) mediating activation of c-jun by BPDE were localized within the promoter region up to -1639, further upstream sequences reduced this transcriptional activation. Thus, when the complete promoter region, up to -4500, was tested, no transcriptional activation was noted following BPDE treatment. These observations suggest that the regulation of c-jun in Clara cells exposed to potent lung carcinogens is mediated at the post-transcriptional level, possibly by reducing the stability and, in turn, the half life of c-jun mRNA. Overall, in contrast to the response of c-jun to numerous carcinogens and stress inducing agents noted in various other cell systems, our findings suggest the existence of a tissue-specific regulatory response for c-jun.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1994

Inhibition of thymidine kinase in cultured mammary tumor cells by the chemopreventive organoselenium compound, 1,4-phenylenebis(methylene)selenocyanate.

To identify mechanisms by which the organoselenium compound 1,4-phenylenebis(methylene)selenocyanate (p-XSC) mediates its chemopreventive activities, we have examined its effects on cell lines derived from breast cancer of humans and rats. When log-phase cells were treated with a dose of 1 microM p-XSC, we observed a significant decrease in thymidine kinase (TK) activity within 4 h, and reduced thymidine incorporation after 24-48 h. When the dose of p-XSC was increased to 2 microM, the decrease in TK was accompanied by a modest, but significant, decrease in thymidine incorporation at 4 h, and a greater inhibition after 24-48 h. At a dose of > or = 3 microM, we observed a large decrease in TK, accompanied by > 70% reduction in thymidine incorporation, as well as decreases in mitochondrial activity and cell numbers, all within 4 h. Equal concentrations of selenium in the form of Na2SeO3 had no effect on the parameters described above. These data suggest that inhibition of thymidine kinase is an early effect of p-XSC in cultured breast tumor cells.

Adenosine Triphosphate↗

PubMed1993

A comparison of techniques for introducing macromolecules into living cells.

Flow cytometry has been used to evaluate several techniques for introducing macromolecules into large numbers of living cells. One technique is cell fusion with red blood cell ghosts loaded with a fluorescent reporter molecule (RBCF). The second technique, termed osmotic lysis of pinosomes (OLP), involves a brief exposure of cells to a hypertonic solution containing the reporter molecule; subsequently, a hypotonic media is added which lyses the pinosomes formed during the hypertonic treatment. A third technique, scrape loading (SL), involves the creation of transient holes in the cell membrane through the application of mechanical forces, which allows for the passage of reporter molecules into cells. A comparison of these techniques is presented here. OLP appears to offer several advantages: It is a simple procedure, virtually all cells are fluorescently labelled, and it is capable of loading larger amounts of material more uniformly into cells while maintaining excellent viability.

Cell Fusion↗

PubMed1993

Expression pattern of proteins that bind to the ultraviolet-responsive element (TGACAACA) in human keratinocytes.

We previously identified an ultraviolet (UV)-responsive element (URE; TGACAACA) that plays a role in both transcription and replication of polyoma sequences. Mouse polyclonal antibodies were raised against affinity-purified URE-bound proteins to characterize their expression patterns. These antibodies specifically recognized two of four URE-bound proteins, of 40 and 68 kDa. The 68-kDa protein was constitutively expressed in human keratinocytes, while the expression of the 40-kDa protein was induced by UV irradiation. Of the two, the 68-kDa protein bound to the URE with greater affinity than the 40-kDa protein, as determined by southwestern analysis. The expression of the 40-kDa protein increased as early as 1 h after UV irradiation of both rat fibroblasts and human keratinocytes and correlated with increased binding to the URE in an electrophoretic mobility shift assay. Other types of damage, as well as heat shock and serum stimulation, also induced the expression of this protein, suggesting that it may play a role in cellular response to stress or damage. The 40-kDa protein was expressed at the highest levels in the S phase of the cell cycle and was induced by aphidicolin, suggesting that it has a role in DNA replication. All together, these results suggest that exposure of human keratinocytes to damage- and stress-inducing agents modulates the expression of proteins that may play a role in regulating cellular response to DNA damage.

Animals↗

PubMed1993

Glycolytic enzymes as DNA binding proteins.

1. Numerous studies have demonstrated the presence of at least four glycolytic enzymes in the nuclear compartment of several cell systems. 2. These include, lactate dehydrogenase, phosphoglycerate kinase, aldolase and glyceraldehyde-3-phosphate dehydrogenase. 3. In some cases the glycolytic enzymes found in the nuclei were a modified form from that found in the cytoplasmic counterpart. 4. In all four cases, the nuclear form of these glycolytic enzymes has been reported to bind DNA. 5. Although none of these enzymes interact with a specific target DNA sequence, their association with DNA may play a role in transcription and replication of DNA through general stabilization of the nuclear matrix or chromatin structure. 6. The present review aims to summarize the current understanding of this phenomenon and to examine the role of the DNA-binding activities of the glycolytic enzymes in cell growth and differentiation.

Animals↗

PubMed1993

General purpose lamps induce polyoma DNA replication in H3 cells.

We have previously demonstrated the ability of UVC (254 nm) radiation to induce asynchronous polyoma replication in rat fibroblast cells (H3 line) that contain an integrated copy of polyoma virus. In the present study we show that general purpose lamps can induce polyoma replication in these cells as well. The amount of UV radiation emitted by three different light sources was determined and the effects of each source on the replication of polyoma DNA was assessed. Our findings indicate that a 100 W incandescent lamp had a minimal effect on replication, whereas a 90 s exposure to a halogen lamp or a 160 W mercury vapor lamp induced replication 1.5-fold and 2-fold, respectively, in comparison with nontreated controls. We have previously shown that asynchronous polyoma replication in H3 cells involves UV-inducible cellular protein factors. Our present results indicate that these factors are also activated by exposure to commonly used lamps that emit comparable doses of UV radiation.

Animals↗

PubMed1993

A transcriptional inhibitor induced in human melanoma cells upon ultraviolet irradiation.

Nuclear proteins from human melanoma cells exhibit strong binding activity to the UV response element (TGACAACA); however, this binding is inhibited following UV-C irradiation. In contrast, the binding of nuclear proteins from rodent fibroblasts and human keratinocytes to the UV-responsive element is initially weak and increases significantly upon UV irradiation. The addition of nuclear proteins from UV-irradiated melanoma cells to those prepared from nonirradiated cells inhibited the binding to the UV-responsive element in a concentration-dependent manner. Fast protein liquid chromatographic analysis of nuclear proteins from UV-irradiated melanoma cells revealed 12 and 14 kilodalton proteins within a fraction which also contained the inhibitory activity. The inhibitor blocks the binding of proteins to three other target sequences, AP1, CREB, and PEBP2, as well as the in vitro transcription of SV40 promoter sequences. The inhibitor was also found in UV-irradiated melanocytes, suggesting that it is tissue specific. The induction of a transcriptional inhibitor in response to UV irradiation represents a regulatory event that may play an important role in the transcriptional response of both normal and malignant melanocytes to UV irradiation.

Base Sequence↗

PubMed1992

Functional role of the ultraviolet light responsive element (URE; TGACAACA) in the transcription and replication of polyoma DNA.

We have previously identified a novel 8 bp sequence (UV-responsive element, URE: TGACAACA) present in the regulatory region of polyoma DNA that interacts with protein factors induced in rat fibroblast cells by exposure to UV light. In the present study, we demonstrate through competitive binding assays that this sequence is distinct from the partially homologous AP1 and CRE target sequences. The proteins that bind to the URE appear to have transcriptional activity in UV-exposed rat fibroblasts. In addition, the URE appears to play a role in promoting the replication of polyoma DNA as determined through two different experimental approaches. Together, these findings suggest that the URE is a novel DNA binding element that interacts with proteins involved in the transcription and replication of polyoma sequences.

Base Sequence↗

PubMed1992

Analysis of mutagenic activity and ability to induce replication of polyoma DNA sequences by different model metabolites of the carcinogenic tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) requires metabolic activation to express its carcinogenic activity. This activation leads to the formation of methylating and pyridyloxobutylating agents. To determine the possible biological effects mediated by each of these metabolic pathways we have studied the activities of model compounds that are metabolized to either a methylating or pyridyloxobutylating species. Each model compound was evaluated for its mutagenic activity in both prokaryotic and eukaryotic cell systems. The model compounds were also tested for their ability to induce asynchronous replication of viral DNA sequences. We demonstrate here that both the methylating model compound acetoxymethylmethylnitrosamine (AMMN) and the pyridyloxobutylating model compound 4-(acetoxymethyl)-1-(3-pyridyl)-1-butanone (NNKOAc) were mutagenic in strains TA98, TA100, and TA1535 but not TA102. While NNKOAc appeared to be 10 times more potent than AMMN in Salmonella, AMMN was found to be a more potent mutagen in mammalian G12 cells. Both chemicals could induce asynchronous replication of polyoma DNA sequences in rat fibroblast cells carrying an integrated copy of the polyoma virus with AMMN appearing to be more active. Measurement of DNA adduct levels suggest that the damage produced by NNKOAc was at least as active as that produced by AMMN when viewed on a per adduct basis. The possible implications of the biological activities exhibited by methylating and pyridyloxobutylating model compounds to NNK induced carcinogenesis are discussed.

Animals↗

PubMed1992

A simple and efficient method for the purification of specific DNA binding proteins.

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DNA↗

PubMed1992

Aldolase-DNA interactions in a SEWA cell system.

In this report we demonstrate the novel finding that aldolase A interacts with DNA sequences in mouse SEWA sarcoma cells. This interaction was initially observed through the identification of a 40 kDa protein which was eluted from a DNA affinity chromatography column consisting of the long terminal repeat (LTR) of the endogenous intracisternal A-type particle (IAP). Microsequencing analysis identified this 40 kDa protein as the glycolytic enzyme, aldolase A. The use of specific anti-aldolase antibodies enabled the identification and subsequent purification of aldolase from the nuclear protein fraction of two SEWA sublines, one that is adherent and one that grows in suspension. In order to confirm our initial finding that aldolase is capable of interacting with DNA, proteins from each subline were immunopurified with anti-aldolase antibodies, eluted and then tested for their ability to interact with IAP-LTR DNA sequences. Interestingly, only aldolase derived from the anchorage dependent SEWA cells was capable of interacting with the IAP-LTR, however, several cell lines derived from human tumors also exhibited this activity. Subsequent studies revealed the ability of aldolase to interact with some but not every DNA sequence tested, implying that there may be a minimal DNA conformation and/or sequence requirement for this activity. The presence of aldolase A in the nuclei and its ability to interact with certain DNA sequences suggest a novel role for this metabolic enzyme.

Animals↗

PubMed1992

Interaction of SEWA sarcoma cell proteins with the intracisternal A-type particle long terminal repeat DNA sequence.

Intracisternal A-type particle (IAP) transcripts are endogenous retrovirus-like sequences expressed during specific stages of normal development and in a variety of murine tumors. In this study, we have analyzed two cell lines derived originally from the SEWA murine osteosarcoma and grown either as ascites or as solid tumors, for proteins that might regulate IAP expression. We found that subline AA7-NA, originally derived from the ascites tumor, expressed about five times more IAP RNA than the AS12-AD subline, which was derived from a solid tumor. In view of this finding, we examined the binding of cellular proteins from the two cell lines to the 5' end of an IAP long terminal repeat sequence. Gel retardation assays of DNA-protein complexes and DNase I footprinting assays identified several DNA sequences within the long terminal repeat fragment that were protected by protein extracts from both SEWA sublines. Gel retardation assays using specific synthetic oligonucleotide sequences that correspond to two of these protected regions revealed different patterns of DNA-protein complexes with extracts from the two SEWA sublines. These data suggest that expression of IAP sequences is regulated by complex mechanisms involving several proteins that appear to differ between the two sublines.

Animals↗

PubMed1992

Evidence that oocyte maturation induced by an oncogenic ras-p21 protein and insulin is mediated by overlapping yet distinct mechanisms.

We have recently shown that a peptide (residues 35-47) from a functional region of the ras p21 protein, thought to be involved in the binding of p21 to GTPase activating protein, the antibiotic azatyrosine, known to induce the ras-recision gene, and the selective protein kinase C inhibitor, CGP 41,251, all inhibit oncogenic p21 protein-induced maturation of oocytes in a dose-dependent manner. We now show that these three agents only partially inhibit insulin-induced oocyte maturation, known to be dependent on activation of cellular p21 protein. On the other hand, the anti-p21 protein antibody Y13-259 completely inhibits both insulin- and oncogenic p21 protein-induced maturation as does a tetrapeptide, CVIM, known to block the enzyme farnesyl transferase which covalently attaches the farnesyl moiety to the p21 protein allowing it to attach to the cell membrane. Our results suggest that while the oncogenic and insulin-activated normal p21 proteins share certain elements of their signal transduction pathways in common, these pathways diverge and allow for selective inhibition of the oncogenic pathway.

Alanine↗