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Colonoscopy is recommended for persons with a familial risk of colorectal cancer (CRC). A familial risk is identified by a family history with CRC and/or predisposing mutation(s). However, such information may not be available. We analysed whether MSI (MicroSatellite Instability) and/or CIN (Chromosome INstability=LOH (loss of heterozygosity) and/or DNA-aneuploidy (abnormal nuclear DNA contents)) could be used as predictors of familial CRC. Formalin-fixed tissue from 97 patients with CRC (29 patients with 2 or more affected first-degree relatives (="cases"), 29 matched CRC controls without a family history, and 39 relatives to cases) were analysed for MSI and CIN. In this small case-control study, no significant differences in the frequencies of MSI and CIN were observed between cases with a family history and their controls without a family history. MSI+;CIN- was observed in 6/29 cases and in 0/29 controls (p=0.02), most frequently in cases with affected siblings, only (3/7). However, for 13 patients from whom several CRC tumours were analysed, concordant results for MSI/LOH/DNA-ploidy were obtained only in 10/9/9. Among cases and relative(s), concordant results for MSI, LOH and DNA-ploidy were obtained in 16/26, 16/26, and 14/25 families, respectively.Although MSI+;CIN- appeared to predict familial CRC with a high specificity, neither MSI, CIN, or MSI+;CIN- are likely to be sufficiently sensitive predictors of familial CRC.

Genotyping of single nucleotide polymorphisms (SNPs) in large populations presents a great challenge, especially if the SNPs are embedded in GC-rich regions, such as the codon 112 SNP in the human apolipoprotein E (apoE). In the present study, we have used immobilized locked nucleic acid (LNA) capture probes combined with LNA-enhancer oligonucleotides to obtain efficient and specific interrogation of SNPs in the apoE codons 112 and 158, respectively. The results demonstrate the usefulness of LNA oligonucleotide capture probes combined with LNA enhancers in mismatch discrimination. The assay was applied to a panel of patient samples with simultaneous genotyping of the patients by DNA sequencing. The apoE genotyping assays for the codons 112 and 158 SNPs resulted in unambiguous results for all patient samples, concurring with those obtained by DNA sequencing.

The expression of CYP1A1 and CYP1B1, encoding enzymes known to play a central role in oxidative metabolism of a wide range of compounds including steroids, was significantly increased in anti-estrogen-resistant human breast cancer cell lines. This was a purely regulatory phenomenon because no gene amplification had occurred. In anti-estrogen-sensitive MCF-7 cells, the steroidal anti-estrogen, ICI 182780, is able to induce the expression of the arylhydrocarbon receptor (AhR)-regulated gene product, CYP1A1, via an estrogen receptor (ER)- mediated process. This observation suggests cross-talk between the AhR and ER systems. Surprisingly, the increased constitutive expression in anti-estrogen-resistant cells of CYP1A1 and CYP1B1 mRNAs, encoding detoxification enzymes, had no effect on the activity of the ICI 182780 compound. The ICI 182780 regulation of estradiol-inducible genes was found to be identical in the resistant and sensitive breast cancer cell lines. In conclusion, anti-estrogen resistance is not due to conversion of ICI 182780 to less active compounds., (Copyright 2000 Wiley-Liss, Inc.)

Insufficient efficacy and/or specificity of antisense oligonucleotides limit their in vivo usefulness. We demonstrate here that a high-affinity DNA analog, locked nucleic acid (LNA), confers several desired properties to antisense agents. Unlike DNA, LNA/DNA copolymers were not degraded readily in blood serum and cell extracts. However, like DNA, the LNA/DNA copolymers were capable of activating RNase H, an important antisense mechanism of action. In contrast to phosphorothioate-containing oligonucleotides, isosequential LNA analogs did not cause detectable toxic reactions in rat brain. LNA/DNA copolymers exhibited potent antisense activity on assay systems as disparate as a G-protein-coupled receptor in living rat brain and an Escherichia coli reporter gene. LNA-containing oligonucleotides will likely be useful for many antisense applications.

Nitric oxide can both stimulate and suppress apoptosis. By reverse transcriptase-polymerase chain reaction and sequencing we show that human breast cancer (MCF-7) cells express endothelial cell nitric-oxide synthase (ecNOS), but not other nitric-oxide synthase isoforms. Inhibition of ecNOS activity in MCF-7 cells increased tumor cell apoptosis, and this effect was also seen following treatment with an NO scavenger. In addition, low concentrations of the NO donor sodium nitroprusside inhibited, whereas high concentrations stimulated MCF-7 cell apoptosis. The ecNOS promoter was found to contain a specific binding site for the apoptosis-regulating protein p53. In co-transfection studies wild-type, but not mutant, p53 down-regulated transcription of an ecNOS promoter-luciferase reporter gene construct. In addition, NO donors up-regulated p53 protein levels in MCF-7 cells. These data point to a previously unrecognized p53-dependent regulation of ecNOS expression that may be important both for regulating apoptosis and for avoiding the generation of genotoxic quantities of NO.

Nitric oxide (NO) is involved in tumor cell apoptosis and has additional effects on tumor blood flow, immune responses, and angiogenesis. We, therefore, studied endothelial cell NO synthase (ecNOS) protein expression in a retrospective series of 118 patients with primary invasive breast cancer. Immunocytochemically stained paraffin sections were used for determining the frequency of (a) tumor cells, (b) intratumoral microvessels, and (c) peritumoral microvessels that were positive for ecNOS. A high density of ecNOS positive microvessels in the normal tissue surrounding the tumor (measured by the variable PEMVD) was associated with significantly better recurrence-free and overall survival. The prognostic significance was observed in a representative series of premenopausal patients and was independent of other factors, including lymph node status. The counting procedure was highly reproducible and correlated to stereological measurements but was influenced by heterogeneity of the tissue samples. Analyzing two sections per patient improved the discriminative power by reducing the influence of tissue heterogeneity and produced highly significant results (recurrence-free survival, P < 0.001; overall survival, P < 0.0001). Immunoreactive ecNOS in microvessels is an independent prognostic factor in breast cancer and may reflect a mechanism of endothelial defense against invasion by tumor cells. Individual variations in ecNOS may be related to environmental, hormonal, and genetic factors and could represent a therapeutic target.

To elucidate the mechanisms involved in anti-oestrogen resistance, two human breast cancer cell lines MCF-7 and the ICI 182780-resistant cell line, MCF-7/182R-6, have been compared with regard to oestrogen receptor (ER) expression, ER function, ER regulation, growth requirements and differentially expressed gene products. MCF-7/182R-6 cells express a reduced level of ER protein. The ER protein is functional with respect to binding of oestradiol and the anti-oestrogens tamoxifen, 4-hydroxy-tamoxifen and ICI 182780, whereas expression and oestrogen induction of the progesterone receptor is lost in MCF-7/182R-6 cells. The ER protein and the ER mRNA are regulated similarly in the two cell lines when subjected to treatment with oestradiol or ICI 182780. Oestradiol down-regulates ER mRNA and ER protein expression. ICI 182780 has no initial effect on ER mRNA expression whereas the ER protein level decreases rapidly in cells treated with ICI 182780, indicating a severely decreased stability of the ER protein when bound to ICI 182780. In vitro growth experiments revealed that the ICI 182780-resistant cell line had evolved to an oestradiol-independent phenotype, able to grow with close to maximal growth rate both in the absence of oestradiol and in the presence of ICI 182780. Comparison of gene expression between the two cell lines revealed relatively few differences, indicating that a limited number of changes is involved in the development of anti-oestrogen resistance. Identification of the differentially expressed gene products are currently in progress.

Effects of transplantable rat insulinomas (IN) and glucagonomas (GLU) on the endogenous pancreas were analyzed using morphometry, immunocytochemistry, in situ hybridization, and staining for apoptotic cells. Hyperinsulinemia (IN-rats) and hyper-GLP-1/glucagonemia (GLU-rats) were both associated with marked islet atrophy (67 and 76% of control average planimetrical islet area, respectively). Selective islet B cell inhibition of proinsulin (I and II) genes as well as of expression of the insulin gene transcription factor, IPF1/STF1, was found in IN-rats. Moreover, these islets were characterized by significant B cells apoptosis in the absence of infiltrating lymphocytes. In GLU-rats selective islet A cell inhibition was observed at the level of glucagon mRNA. These islets contained small, highly condensed but clearly active B cells with prominent IPF1/STF1-positive nuclei, surrounded by densely packed glucagon-negative cells with reduced cytoplasm. Furthermore, an active apoptotic process was found exclusively in the exocrine pancreas of GLU-rats. Thus, in IN-rats, islet B cell mass reduction is distinguished by non-immune-mediated programmed cell death, while GLU-rats exhibit A cell mass reduction by cytoplasmic retraction and selective exocrine apoptosis.

Activation of the protein kinase C signaling pathway by tumor-promoting phorbol esters, such as 4 beta-phorbol 12-myristate 13-acetate (PMA), induced a decrease in the level of p53 mRNA in several serum-starved human cell lines. Also, the tumor-promoting phosphatase inhibitor okadaic acid induced a decrease in the p53 mRNA level in the cell lines. Normal diploid as well as various tumor cell lines were tested. Two tumor cell lines, HeLa and A549, both containing the wild-type p53 gene, but very different levels of p53 protein, were studied in detail. In both cell lines, the level of p53 mRNA was minimal after 9 h of exposure to PMA. After approximately 120 h, the p53 mRNA level was similar to the pretreatment level. PMA induced a similar transient decrease in the level of p53 protein in the A549 cell line. The decrease in the p53 mRNA level could not be explained by changes in the transcriptional rate or the p53 mRNA stability. The protein synthesis inhibitor cycloheximide completely abolished the PMA-induced down-modulation of the p53 mRNA, suggesting that a short-lived protein was involved in the down-modulation. Flow cytometric cell cycle analysis showed that the phorbol ester treatment induced a block in the late G1 phase. The blockage was transient, and its duration correlated with the level of p53 protein in the two cell lines. We propose that the protein kinase C-catalyzed phosphorylation of p53 may be a key event in the down-modulation of p53 expression as well as in the induced blockage of the cell cycle.

The sialosyl Lewis a antigen was identified in a ganglioside extract from a malignant human urothelial cell line (Hu 1703He) by fast atom bombardment mass spectrometry. The presence of this antigen in urothelial cell lines with varying tumorigenic properties was further studied using the 19-9 monoclonal antibody (MAb). The sialosyl Lewis a ganglioside was expressed only in the tumorigenic cell lines. Thus, the expression of this antigen is a marker of malignancy for human urothelial cell lines. Mass spectrometry also suggests that the fucosyl GM1 ganglioside was expressed in the Hu 1703He cell line. This was confirmed using the F12 MAb, specific for fucosyl GM1. However, the expression of this antigen was not confined to cell lines with tumorigenic properties.

The expression of a number of cellular oncogenes was investigated in human urothelial cell lines with different in vitro growth properties. Constitutively elevated levels of expression of c-myc RNA were found in Hu609, an immortalized, nontumorigenic cell line that was derived from normal urothelium, and in the bladder carcinoma cell line T24. Potential mechanisms that might underlie deregulation of c-myc expression in these cells were investigated. It was found that the c-myc gene was apparently intact and not amplified in Hu609 and T24. No increased stability of c-myc RNA was detected. A c-myc-CAT fusion construct containing 2.5 kb of normal c-myc 5' sequences showed levels of expression that paralleled the overexpression of the endogenous gene, indicating that the high constitutive levels of c-myc expression were due, at least in part, to alterations in the activities of cellular trans-acting transcriptional regulators.

Three strong and two minor rpsA promoters were found by nuclease S1 mapping, promoter cloning and in vitro transcription. The longest transcript encodes a protein, located upstream from rpsA with a molecular weight of 25,000. The identity of this protein remains to be established. The other rpsA promoters are located within the gene for this 25 K protein. The rpsA leader region including the sequence of the 25 K protein and its promoter was DNA sequenced.

In a previous study we have shown that established human urothelial cell lines, representing grade of transformation II (TGr II, non-tumorigenic, non-invasive cells), are characterized by accumulation of the GM2 ganglioside as compared to cell lines of TGr III with tumorigenic and invasive properties. In the present study, the analysis of gangliosides from two tumorigenic sublines obtained after transfection of the TGr II cell line HCV 29 with the v-raf-oncogene, provided further evidence for the inverse relationship between tumorigenicity and the GM2 ganglioside expression. The two transfected sublines: T112C1 and T112D1, representing TGr III, were characterized by a decreased level of GM2 which was accompanied by an increased content of the GM3 ganglioside as compared to the parental HCV 29 cell line.

The effect of a single treatment with the skin tumor-promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on the expression of the cellular proto-oncogenes, c-myc, c-rasHa, c-rasKi and c-fos was examined in the non-tumorigenic human bladder epithelial cell line HCV 29. TPA (1 microgram/ml) increased the transcription of the c-fos gene of HCV 29 at least 50-fold, and this stimulation was observed within minutes. The response was transient, and was accompanied by a rapid and transient change in cell morphology. The expression of c-myc, c-rasHa and c-rasKi were not enhanced by the TPA treatment. These results show that human bladder epithelial cells respond to a known skin tumor-promoter, TPA, by altering the transcription of a specific proto-oncogene in these cells.

The present study was undertaken to determine the feasibility of using in situ hybridization techniques to identify oncogene transcription in cultured cells. Following in situ hybridization with 32P-labeled v-src and v-Ha-ras DNA probes, src and Ha-ras related transcripts were identified in cell lines transfected with v-src and Ha-ras, respectively. In both the v-src and c-Ha-ras transfected cell lines, the number of silver grains over individual cells were significantly higher (p less than 0.001, t-test) than in a non-transfected, non-tumorigenic, rat esophageal epithelial cell line. There was a highly variable number of silver grains above individual cells. Significantly fewer silver grains were counted over cells that had been preincubated with either non-labeled v-src or v-Ha-ras DNA or that were pretreated with RNase A. Both oncogene transfected cell lines contained approximately 10 times more oncogene related mRNAs than non-transfected cells as judged by the numbers of silver grains over individual cells. Filter-hybridization analysis of the transfected and non-transfected cell lines confirmed that the expression of src and Ha-ras transcripts was higher in the transfected cell lines than in the non-transfected cell line. Therefore, the in situ hybridization technique would appear useful for the identification of oncogene transcripts in single cells and could potentially be applied to cytological preparations of human cells and to human tumor cells in culture.

Transfection of the v-raf oncogene into immortalized, nontumorigenic human bladder epithelial cells resulted in the isolation of two tumorigenic transformants. Both were identified as human and of the same origin as the parent cell line by human leukocyte antigen typing and Southern blot analysis. Both the primary tumorigenic transfectants and the cell lines established from the induced tumors expressed v-raf mRNA and v-raf protein. In both tumorigenic transformants the level of c-myc mRNA was enhanced compared with that of the parent cell line.

The effect of the skin tumor-promoter TPA (12-O-tetradecanoylphorbol-13-acetate) on expression of cellular proto-oncogenes has been examined in cell lines derived from human urothelium. A single treatment with TPA (1 microgram/ml) increased the transcription of c-fos and c-myc proto-oncogenes at least 20-fold in the mortal cell line HU 1752. The induction was transient and was accompanied by a rapid but transient change in cell morphology. When immortalized cell lines were treated with TPA a similar rapid and transient morphological response was observed, but the TPA treatment only increased the level of c-fos mRNA, suggesting that the normal regulation of c-myc transcription is altered in immortalized cells irrespective of their tumorigenic properties. The levels of c-Ha-ras and c-Ki-ras mRNAs were unaffected by TPA treatment in all cell lines.