RINF (CXXC5) is overexpressed in solid tumors and is an unfavorable prognostic factor in breast cancer.

BACKGROUND: We have previously described the essential role of the retinoid-inducible nuclear factor (RINF) during differentiation of hematopoietic cells and suggested its putative involvement in myeloid leukemia and preleukemia. Here, we have investigated whether this gene could have a deregulated expression in malignant tissues compared with their normal tissues of origin and if this potential deregulation could be associated with important clinicopathological parameters. PATIENTS AND METHODS: RINF messenger RNA expression was examined in biopsies from locally advanced breast tumors, metastatic malignant melanomas, and papillary thyroid carcinomas and compared with their paired or nonpaired normal reference samples. Further, the prognostic role of RINF expression was evaluated in locally advanced breast cancer. RESULTS: RINF expression was significantly higher in all tumor forms (primary breast, and thyroid cancers and metastatic melanomas) as compared with normal control tissues (P < 0.001 for each comparison). Importantly, high levels of RINF expression correlated to a poor overall survival in breast cancer (P = 0.013). This finding was confirmed in three independent public microarray datasets (P = 0.043, n = 234; P = 0.016, n = 69; P = 0.001, n = 196) and was independent of tamoxifen therapy. Notably, high levels of RINF was strongly associated with TP53 wild-type status (P = 0.002) possibly indicating that high levels of RINF could substitute for TP53 mutations as an oncogenic mechanism during the malignant development of some cases of breast cancer. CONCLUSIONS: Our data indicate that (i) RINF overexpression is associated with the malignant phenotype in solid tumors and (ii) RINF overexpression represents an independent molecular marker for poor prognosis in breast tumors.

The presence of multiple c-type cytochromes at the surface of the methanotrophic bacterium Methylococcus capsulatus (Bath) is regulated by copper.

Identification of surface proteins is essential to understand bacterial communication with its environment. Analysis of the surface-associated proteins of Methylococcus capsulatus (Bath) revealed a highly dynamic structure responding closely to the availability of copper in the medium in the range from approximately 0 to 10 microM. Several c-type cytochromes, including three novel multihaem proteins, are present at the cellular surface, a feature that is otherwise a peculiarity of dissimilatory metal-reducing bacteria. At low copper concentrations, the cytochrome c(553o) and the cytochrome c(553o) family protein, encoded by the MCA0421 and MCA0423 genes, respectively, are major constituents of the surfaceome and show a fine-tuned copper-dependent regulation of expression. Two novel members of the cytochrome c(553o) family were identified: MCA0338 was abundant between 5 and 10 microM copper, while MCA2259 was detected only in the surface fraction obtained from approximately 0 microM copper cultures. The presence at the bacterial surface of several c-type cytochromes, generally involved in energy transduction, indicates strongly that redox processes take place at the bacterial surface. Due to the unique role of copper in the biology of M. capsulatus (Bath), it appears that c-type cytochromes have essential functions in copper homeostasis allowing the cells to adapt to varying copper exposure.

Breast cancer prognostication and prediction in the postgenomic era.

Expanding knowledge, together with implementation of new techniques, has fuelled the area of translational medical research aiming at improving prognostication as well as prediction in cancer therapy. At the same time, new discoveries have revealed a biological complexity we were unaware of only a decade ago. Thus, we are faced with novel challenges with respect to how we may explore issues such as prognostication and predict drug resistance in vivo. While microarray analysis exploring expression of thousands of genes in concert represents a major methodological advancement, discoveries such as the finding of different mechanisms of epigenetic silencing, intronic mutations, that most gene transcripts in the human genome are subject to alternative splicing and that hypersplicing seems to be a tumour-related phenomenon, exemplifies a complex pathology that may not be explored with use of single analytical methods only. This paper discusses clinical settings for studying drug resistance in vivo together with a discussion of contemporary biology in this field. Notably, each individual parameter which has been found correlated to drug resistance in vivo so far represents either a direct drug target or a factor involved in DNA repair or apoptosis. On the basis of these findings, we suggest drug resistance may be explored on the basis of upfront biological hypotheses.

Induction of apoptosis in human cells by RNAi-mediated knockdown of hARD1 and NATH, components of the protein N-alpha-acetyltransferase complex.

Protein N-epsilon-acetylation is recognized as an important modification influencing many biological processes, and protein deacetylase inhibitors leading to N-epsilon-hyperacetylation of histones are being clinically tested for their potential as anticancer drugs. In contrast to N-epsilon-acetyltransferases, the N-alpha-acetyltransferases transferring acetyl groups to the alpha-amino groups of protein N-termini have only been briefly described in mammalians. Human arrest defective 1 (hARD1), the only described human enzyme in this class, complexes with N-acetyltransferase human (NATH) and cotranslationally transfers acetyl groups to the N-termini of nascent polypeptides. Here, we demonstrate that knockdown of NATH and/or hARD1 triggers apoptosis in human cell lines. Knockdown of hARD1 also sensitized cells to daunorubicin-induced apoptosis, potentially pointing at the NATH-hARD1 acetyltransferase complex as a novel target for chemotherapy. Our results argue for an essential role of the NATH-hARD1 complex in cell survival and underscore the importance of protein N-alpha-acetylation in mammalian cells.

Gene expression profile in oral squamous cell carcinomas and matching normal oral mucosal tissues from black Africans and white Caucasians: the case of the Sudan vs. Norway.

Expression profile of 588 known genes relating to tumour biology, was examined between oral squamous cell carcinomas (OSCCs) and matching normal oral mucosal tissues (NOMTs) obtained from Sudanese (n=11) and Norwegian (n=11) patients. cDNA probes were synthesised from total RNA and hybridised with the Atlas human cancer cDNA expression array membranes. RT-PCR and immunohistochemistry were applied to confirm the expression pattern of a subset of the 588 genes. Differences in expression of the genes examined were found between the OSCCs and the NOMTs on the Atlas membranes. Several of these genes were either up- or down-regulated 1.6-fold or higher in the OSCCs compared to the NOMTs in the cases from the two populations. We found that 181 (31%) and 195 (33%) genes were either up-regulated or down-regulated in the OSCCs from the Sudan and Norway, respectively. From the total number of genes (n=376) found expressed in the OSCCs investigated from the two countries, 53 genes (14%) showed common expression profile [35 (66%) were up-regulated and 18 (34%) were down-regulated] and 70 genes (19%) showed opposite regulation status. Results of the RT-PCR and immunohistochemistry confirmed the hybridisation data. These findings may provide an OSCCs-specific gene expression profile in patients from the two countries, suggesting that alterations of 123 genes are common in these OSCCs regardless of ethnic differences or other socio-cultural risk factors between the patients from the two countries. The findings might further suggest that specific genes are frequently involved in these OSCCs, which may provide novel clues as diagnostic, prognostic biomarkers and/or targets for therapy. The Atlas human cancer cDNA expression array technique can be useful to examine and describe the expression profile of known genes frequently involved in OSCCs from different populations.

Establishment of okadaic acid resistant cell clones using a cDNA expression library.

The mechanism whereby the universal apoptogen and serine/threonine phosphatase inhibitor okadaic acid (OA) kills cells, is still unclear. To create a novel tool for probing of OA action, fibroblasts were selected for OA-resistance after infection with a retroviral Jurkat T-cell cDNA expression library. Twenty-one clones were selected. Two of these (OAR1, OAR2) were studied in detail. OAR1 and 2 had each a retrovirally introduced short cDNA, corresponding to a human gene (oar1 and oar2, respectively) with unknown function. Reintroduction of oar1 or oar2 cDNA into wild-type cells reproduced the OA-resistant phenotype. OAR1 and 2 were cross-resistant to other phosphatase inhibitors (calyculin A, cantharidin), but not to staurosporine or microinjected Cytochrome c, thus, indicating a disturbance in a limited number of death pathways, upstream or independent of apaf-1/caspases-3/9. The action of OA involved caspase-dependent and caspase-independent components. Both components were less efficient in OAR1 and 2, than in wild-type cells. Subtle differences existed between OA-induced phosphoprotein patterns in wild-type cells, OAR1, and OAR2, indicating that a narrow selection of protein phosphorylation events had been targeted. We propose that the clones have defects in a hitherto non-elucidated signal pathway linking OA-induced protein phosphorylation to initiation of a death execution pathway provided with a caspase-dependent amplification loop. The novel OA-resistant cell clones will be used to elucidate the significance for apoptosis of oar1 and 2, their link to altered protein phosphorylation, and the potential link of the latter to initiation of apoptosis.

Difference in patterns of Met expression in papillary thyroid carcinomas and nonneoplastic thyroid tissue.

Met protein is a tyrosine kinase receptor for hepatocyte growth factor (HGF). c-Met has morphogenic, mitogenic, and motogenic properties and is overexpressed in many solid tumors. We studied c-met mRNA and protein expression in papillary thyroid carcinomas and nonneoplastic thyroid tissue. The c-met mRNA was detected in all biopsies by reverse transcriptase-polymerase chain reaction and by hybridization of complex cDNA probes to a c-met-specific DNA fragment in a dot blot array. Immunohistochemistry on fresh frozen biopsies showed Met protein localized along the basal cell membrane of normal thyrocytes in 32 of 35 nonneoplastic thyroid tissue specimens, sometimes associated with weak cytoplasmic reactivity but without apical cell membrane staining. In papillary carcinomas an increased Met protein expression was seen, comprising a cytoplasmic (33 of 49) and apical cell membrane (24 of 49) immunoreactivity, whereas only 1 of 49 biopsies showed basal cell membrane staining. A 145-kDa Met-specific band was detected by Western immunoblotting on protein extracts from papillary carcinomas. The tight junction protein zona occludens-1 (ZO-1), studied by immunohistochemistry, was weakly expressed along the apical cell membrane in 10 nonneoplastic biopsies. In contrast, increased and cytoplasmic/apical membranous ZO-1 immunostaining was seen in 11 of 15 papillary carcinomas. Nuclear ZO-1 staining was present in a few papillary carcinomas with partial dedifferentiation. The concomitant overexpression and subcellular redistribution of Met and ZO-1 proteins indicate a change in cell polarity in papillary carcinomas compared to nonneoplastic thyroid tissue. These observations may reflect an important feature of the tumorigenesis of papillary thyroid carcinomas. No significant association was found between semiquantitative immunohistochemical assessment of Met protein and clinical parameters in papillary carcinoma patients.

Influence of TP53 gene alterations and c-erbB-2 expression on the response to treatment with doxorubicin in locally advanced breast cancer.

TP53 status [mutations, immunostaining, and loss of heterozygosity (LOH)], expression of c-erbB-2, bcl-2, and histological grading were correlated to the response to doxorubicin monotherapy (14 mg/m2) administered weekly to 90 patients with locally advanced breast cancer. Mutations in the TP53 gene, in particular those affecting or disrupting the loop domains L2 or L3 of the p53 protein, were associated with lack of response to chemotherapy (P = 0.063 for all mutations and P = 0.008 for mutations affecting L2/L3, respectively). Similarly, expression of c-erbB-2 (P = 0.041), a high histological grade (P = 0.023), and lack of expression of bcl-2 (P = 0.018) all predicted chemoresistance. No statistically significant association between either p53 immunostaining or TP53 LOH and response to therapy was recorded, despite the finding that both were associated with TP53 mutation status (p53 immunostaining, P < 0.001; LOH, P = 0.021). Lack of immunostaining for p53 despite mutation of the TP53 gene was particularly seen in tumors harboring nonsense mutations or deletions/splices (7 of 10 negative for staining compared with 4 of 16 with missense mutations). TP53 mutations (total/affecting L2/L3 domains) were associated with expression of c-erbB-2 (P < 0.001 for both), high histological grade (P = 0.001 and P = 0.025), and bcl-2 negativity (P = 0.003 and P = 0.002). TP53 mutations, histological grade, and expression of bcl-2 (but not LOH or c-erbB-2 expression) all predicted for relapse-free as well as breast cancer-specific survival in univariate analysis (Ps between <0.0001 and 0.0155), but only tumor grade was found to be predictive in multivariate analysis (P = 0.01 and P = 0.0007, respectively). Our data are consistent with the hypothesis that certain TP53 mutations predict for resistance to doxorubicin in breast cancer patients. However, the observation that the majority of patients with TP53 mutations affecting or disrupting the L2/L3 domains with LOH in addition (n = 12) obtained a partial response (n = 4) or stabilization of disease (n = 5) during chemotherapy suggests redundant mechanisms to compensate for loss of p53 function. Our findings are consistent with the hypothesis that other defects may act in concert with loss of p53 function, causing resistance to doxorubicin in breast cancers.

Expression and alternative splicing of c-ret RNA in papillary thyroid carcinomas.

Somatic rearrangements of the ret receptor tyrosine kinase have been consistently reported in papillary thyroid carcinomas (PTC). It is unclear whether the expression of wild-type c-ret may also be implicated in thyroid tumorigenesis. We studied ret mRNA expression in PTC from Norwegian patients. Using RT-PCR, wild-type ret mRNA was detected in all of 22 PTC and in a PTC cell line. c-ret mRNA was clearly overexpressed in PTC as compared to non-neoplastic thyroid tissue. Hybridization using ret exon DNA dot blot arrays and complex cDNA probes confirmed expression of ret RNA in thyroid biopsies. In accordance with the RNA data, Western immunoblotting showed evidence of wild-type Ret protein in PTC. Rearrangements generating the ret/PTC oncogenes co-existed with c-ret mRNA in PTC. Multiple alternative ret splicing variants were detected in PTC. Four novel ret splicing events were found in the region encoding the extracellular domain. The open reading frames of these transcripts were all in-frame with the Ret tyrosine kinase domain. In the central ret mRNA region encoding the cysteine-rich, transmembrane, and main tyrosine kinase domains, no evidence of alternative splicing was detected. Two alternative splice events were detected in the ret mRNA encoding the C-terminal part of Ret protein harboring tyrosine residues important for Ret signaling, excluding exon 19, or retaining intron 19, respectively. Ribonuclease protection assays confirmed the presence of ret alternative splicing events in thyroid biopsies. We conclude that in addition to ret/PTC rearrangements, wild-type c-ret mRNA and alternatively spliced ret transcripts are present in PTC. Transcriptional up-regulation and post-transcriptional mechanisms of c-ret RNA processing may contribute to differences in expression of Ret protein observed in PTC compared to non-neoplastic thyroid tissue.

Expression of a peptide binding to receptor for activated C-kinase (RACK1) inhibits phorbol myristoyl acetate-stimulated phospholipase D activity in C3H/10T1/2 cells: dissociation of phospholipase D-mediated phosphatidylcholine breakdown from its synthesis.

The C3H/10T1/2 Cl8 HAbetaC2-1 cells used in this study express a peptide with a sequence shown to bind receptor for activated C-kinase (RACK1) and inhibit cPKC-mediated cell functions. Phorbol myristoyl acetate (PMA) strongly stimulated phosphatidylcholine (PtdCho)-specific phospholipase D (PLD) activity in the C3H/10T1/2 Cl8 parental cell line, but not in Cl8 HAbetaC2-1 cells, indicating that full PLD activity in PMA-treated Cl8 cells is dependent on a functional interaction of alpha/betaPKC with RACK1. In contrast, the PMA-stimulated uptake of choline and its subsequent incorporation into PtdCho, were not inhibited in Cl8 HAbetaC2-1 cells as compared to Cl8 cells, indicating a RACK1-independent but PKC-mediated process. Increased incorporation of labelled choline into PtdCho upon PMA treatment was not associated with changes of either CDP-choline: 1,2-diacylglycerol cholinephosphotransferase activity or the CTP:phosphocholine cytidylyltransferase distribution between cytosol and membrane fractions in Cl8 and Cl8 HAbetaC2-1 cells. The major effect of PMA on the PtdCho synthesis in C3H/10T1/2 fibroblasts was to increase the cellular uptake of choline. As a supporting experiment, we inhibited PMA-stimulated PtdH formation by PLD, and also putatively PtdH-derived DAG, in Cl8 cells with 1-butanol. Butanol did not influence the incorporation of [(14)C]choline into PtdCho. The present study shows: (1) PMA-stimulated PLD activity is dependent on a functional interaction between alpha/betaPKC and RACK1 in C3H/10T1/2 Cl8 fibroblasts; and (2) inhibition of PLD activity and PtdH formation did not reduce the cellular uptake and incorporation of labelled choline into PtdCho, indicating that these processes are not directly regulated by PtdCho-PLD activity in PMA-treated C3H/10T1/2 Cl8 fibroblasts.

Differential TPA and PDGF-BB effects on subcellular localisation of PKC alpha and beta I in C3H/10T1/2 cells.

60-75% of PKC alpha and 30-40% of PKC beta I++ protein was located to the membranes and nuclear/nuclear associated endoplasmatic reticulum fractions of resting 10T1/2CI 8 mouse embryo fibroblasts. On average, 35% of the PKC alpha and 65% of the PKC beta I++ isoforms existed in a soluble state. Maximum PDGF-BB-mediated Erk1 activation was obtained without significant changes in soluble PKC alpha- or PKC beta I++- levels. The subcellular localisation of PKC alpha and PKC beta I was not affected by PDGF-BB treatment. 12-O-tetradecanoyl phorbol-13-acetate (TPA) caused translocation of cytoplasmic PKC alpha and beta I to the nucleus/nuclear associated endoplasmatic reticulum fraction. Down-regulation of PKC and bisindolylmaleimide I inhibited both TPA and PDGF-BB stimulated Erk1 activity. We are the first to show that PDGF-mediated activation of Erk1 involves a PKC-dependent step in 10T1/2CI 8 cells. We also provide novel evidence that PDGF-BB mediated Erk1 activation can take place in these cells without apparent recruitment of soluble PKC alpha/beta I to the particulate cell fractions.

Expression of heregulins and associations with the ErbB family of tyrosine kinase receptors in papillary thyroid carcinomas.

Heregulin (Hrg) growth factors are natural ligands for ErbB3 and ErbB4. Because these receptors are involved in papillary thyroid carcinomas, we studied expression of Hrgs in fresh-frozen thyroid tissue and analyzed for possible coexpressions among the 4 members of the ErbB family of growth factor receptors and Hrgs in papillary carcinomas. Immunohistochemistry for the Hrg precursor isoform (134 biopsies from 101 patients) showed nuclear immunostaining in 83% of papillary carcinomas but not in normal thyroid tissue. Cytoplasmic immunopositivity for the Hrg precursor isoform was moderate or strong in 78% of papillary carcinoma specimens and weak in 13% of normal thyroid tissue samples. Western blot for the Hrg precursor isoform showed the expected protein band of approximately 70 kDa in papillary carcinomas, but not in non-neoplastic thyroid biopsies. Whereas weak cytoplasmic immunostaining for the mature Hrg alpha, beta1, and beta3, was present in 48, 38, and 51% of papillary carcinomas, respectively, normal thyroid tissue samples were negative. Hrg mRNA was present in both tumor and nontumor tissue, with evidence of increased mRNA expression in 5 of 12 papillary carcinomas. RT-PCR of hrg mRNA, with subsequent DNA sequencing, confirmed the presence of hrg alpha, beta1, beta2, and beta3 mRNA in papillary carcinomas. In 55 papillary carcinomas, increased cytoplasmic immunostaining of the ErbB2 and ErbB3 receptors was significantly associated with each other and with cytoplasmic epidermal growth factor receptor (EGFR) immunoreactivity, indicating a common regulatory mechanism. Cytoplasmic staining for Hrg beta3 was significantly associated with ErbB3 immunostaining, indicating this receptor as the cognate one. The overexpression and nuclear localization of the Hrg precursor isoform were not associated with the expression of ErbB-receptors. This may reflect an unknown mechanism of action, possibly independent of the ErbB receptor system.

A mutated PML/RARA found in the retinoid maturation resistant NB4 subclone, NB4-R2, blocks RARA and wild-type PML/RARA transcriptional activities.

The fusion protein PML/RARA, associated with acute promyelocytic leukemia behaves as an abnormal retinoic acid (RA) receptor with altered transactivation properties but is still inducible by RA. The chimeric protein is thought to promote leukemogenesis but also paradoxically to mediate the sensitivity to ATRA of APL cells. This has been supported by works reporting that in vitro ATRA resistance is characterized by defects in the RARA/E-domain of PML/RARA. In the present report, we identified a new mutation in the E domain of PML/RARA which is associated with a RA-resistant subline of NB4 cells; NB4-R2. This mutation, identical to the Gln411 mutation found in HL60-R, changes the amino acid Gln903 to an in-phase stop codon, generating a truncated form of PML/RARA which has lost 52 amino acids at its C-terminal end. We have studied the effect of the truncated PML/RARA protein on PML NB formation and RARA and PML/RARA transcriptional activity. We show here that the fusion mutant exerts a dominant negative effect on wild-type PML, PML/RARA and RARA transcription activity. These findings highlight the important role of the RARA E-domain of PML/RARA in mediating RA sensitivity in APL cells.

Ectopic expression of Bcl-2 switches over nuclear signalling for cAMP-induced apoptosis to granulocytic differentiation.

The IPC-81 myeloid leukaemia cells undergo apoptosis rapidly after cAMP stimulation (6 h) and cell death is prevented by early over-expression of the cAMP-inducible transcription repressor ICER, that blocks cAMP-dependent nuclear signalling. Therefore, the expression of specific genes controlled by CRE-containing promoters is likely to determine cell fate. We now show that cAMP-induced cell death also is abrogated by the over-expression of the anti-apoptotic gene, Bcl-2. Contrary to ICER, Bcl-2 does not affect cAMP-signalling and allows the analysis of cAMP responses in death rescued cells. The Bcl-2 transfected cells treated with 8-CPT-cAMP were growth-arrested and thereafter cells embarked in granulocytic differentiation, with no additional stimulation. Neutrophilic polynuclear granulocytes benefited from a long life span in G0-G1 and remained functional (phagocytosis). This work demonstrates that, using anti-apoptosis regulators, 'death signals' could be exploited to trigger distinct biological responses. Indeed, cAMP signal can trigger several simultaneously developing biological programs, in the same cell, i.e., growth regulation, apoptosis and differentiation. This cell system should prove useful to determine how a tumour cell can be re-programmed for either apoptosis or functional maturation by physiological signals.

RAR-independent RXR signaling induces t(15;17) leukemia cell maturation.

Although retinoic acid receptor alpha (RARalpha) agonists induce the maturation of t(15;17) acute promyelocytic leukemia (APL) cells, drug treatment also selects leukemic blasts expressing PML-RARalpha fusion proteins with mutated ligand-binding domains that no longer respond to all-trans retinoic acid (ATRA). Here we report a novel RARalpha-independent signaling pathway that induces maturation of both ATRA-sensitive and ATRA-resistant APL NB4 cells, and does not invoke the ligand-induced alteration of PML-RARalpha signaling, stability or compartmentalization. This response involves a cross-talk between RXR agonists and protein kinase A signaling. Our results indicate the existence of a separate RXR-dependent maturation pathway that can be activated in the absence of known ligands for RXR heterodimerization partners.

Enhanced expression of EGF receptor and low frequency of ras mutations in X-ray-induced rat thyroid tumours.

Radiation is recognized as a carcinogenic factor for the thyroid gland. In this experimental study, oncogene expression was investigated in radiation-induced rat thyroid tumours. Forty 3-month-old Wistar rats received X-ray-irradiation to the neck region; 40 animals were untreated controls. After 14 months, thyroid tumours had developed in 25 of the 29 irradiated animals still alive; 76% of these tumours were considered malignant. No tumours developed in controls. Mutations of codons 12-13 and 59-63 of H-, K- and N-ras were analysed by PCR-SSCP (single-strand conformation polymorphism analysis) and sequencing of DNA from thyroid tissue. SSCP indicated a ras mutation frequency of 8%, but only one K-ras codon 12 (Gly-Cys) mutation was confirmed by sequencing. Protooncogene expression was analysed by mRNA slot blot hybridization analysis and immunohistochemistry. K-ras mRNA expression and EGF receptor mRNA and protein expression were significantly increased in the irradiated animals compared with controls, and in tumours versus nontumour tissue. This study of radiation-induced rat thyroid tumours demonstrates that ras expression may be subject to changes apart from activating mutations. Increased expression of EGF receptor in the tumours parallels the situation in human thyroid cancer.

Mutations of the p53 gene in oral squamous-cell carcinomas from Sudanese dippers of nitrosamine-rich toombak and non-snuff-dippers from the Sudan and Scandinavia.

Using PCR-SSCP/DNA sequencing methods, we analyzed 14 oral squamous-cell carcinomas (OSCCs) and 8 pre-malignant oral lesions from different Sudanese patients for prevalence of mutations in exons 5 to 9 of the p53 gene in relation to toombak-dipping status. OSCCs (14 from Sudan, 28 from Scandinavia), and 3 pre-malignant oral lesions from Sudanese non-dippers were used as controls. A statistically significant increased incidence in mutations of the p53 gene was found in OSCCs from toombak dippers (93%; 13/14), as compared with those from non-dippers in Sudan (57%; 8/14) and in Scandinavia (61%; 17/28) respectively. In OSCCs from dippers, mutations were found in exons 5 to 9, while in those from non-dippers they were found in exons 5, 7, 8, 9, and no mutations were found in exon 8 in any of the OSCCs from Sudan. Certain types of mutations, however, were similar with respect to exposure to toombak. OSCCs from dippers showed 15 transversions, 9 transitions, 3 insertions and one deletion, compared with 7 transversions, 2 transitions and one deletion found in OSCCs from Sudanese non-dippers, and 9 transversions, 17 transitions and 2 insertions found in those from non-dippers in Scandinavia. No mutations were found in any of the non-malignant oral lesions in relation to dipping or non-dipping status. These findings suggest that (i) the use of toombak plays a significant role in induction of increased p53 gene mutations, (ii) mutations observed were similar to those induced by tobacco-specific N-nitrosamines (TSNAs) in experimental animal models and those already reported in toombak dippers, (iii) types of mutations associated with TSNAs were similar in the exposed and the control groups, (iv) a novel mutation in exon 6 was found in the OSCCs from toombak dippers, (v) the p53 exons 5 (codon 130), 6 (codons 190, 216) and 7 (codons 229, 249, 252) mutations are probable hot spots for toombak-related OSCCs. Further studies are necessary to validate the increased incidence and exon locations of the p53-gene mutations as a biomarker of malignant transformation in populations in which the oral use of tobacco is habitual.

Expression of biomarkers (p53, transforming growth factor alpha, epidermal growth factor receptor, c-erbB-2/neu and the proliferative cell nuclear antigen) in oropharyngeal squamous cell carcinomas.

Using immunohistochemistry, expression of p53, transforming growth factor-alpha (TGF-alpha), epidermal growth factor receptor (EGFR), c-erbB-2/neu and proliferating cell nuclear antigen (PCNA) was examined in 26 fresh frozen tissue specimens of oropharyngeal squamous cell carcinomas (SCCs). p53 gene mutations were examined by polymerase chain reaction (PCR)/DNA sequencing methods in 22 carcinomas. The findings were examined for correlations with patients' clinicopathological parameters. Expressions of p53 and PCNA were also examined in 21 formalin-fixed corresponding tissues. Of the fresh frozen tissue specimens, 77% (20/26) showed expression and 68% (15/22) showed mutations (substitutions) of the p53, with significant clustering of the mutations in exons 5 (8/22; 36%), 7 (4/22; 18%) and 8 (5/22; 23%). No mutations were found in exon 6. There was a discordance between expression of p53 protein and mutations of the gene. Parallel to expression and mutations of the p53 found in most of the specimens, expression of TGF-alpha, EGFR, c-erbB-2/neu and PCNA was found in 88% (22/25), 92% (23/25), 58% (14/24) and 91% (21/23) of the specimens, respectively. For the formalin-fixed tissue specimens, 62% (13/21) and 90% (19/21) expressed p53 and PCNA, respectively. Examining for correlations with patients' clinicopathological parameters, expression of p53, TGF-alpha, EGFR and c-erB-2/neu seemed to negatively correlate with the increase of the tumour grade. The present work suggests that: (1) lack of negative growth regulation due to inactivation of the p53 gene together with activation of other proto-oncogenes are necessary genetic events in the carcinogenesis of oropharyngeal SCCs; (2) in oropharyngeal SCCs, p53 gene mutations were clustered in exons 5 (codons 130-186), 7 (codons 230-248) and 8 (codons 271-282) which perhaps suggests that tobacco carcinogens probably affect the mutational hot spots of the p53 gene at codons 157, 175, 186, 248, 273 and 282; and (3) fresh frozen and formalin-fixed tissue specimens give similar results when an immunohistochemical method is applied. The importance of p53, TGF-alpha, EGFR, c-erbB-2/neu and PCNA as biomarkers in oropharyngeal SCCs deserves particular attention because it might offer further understanding of the development of these carcinomas.

Choline derived from the phosphatidylcholine specific phospholipase D is not directly available for the CDP choline pathway in phorbol ester-treated C3H10T1/2 Cl 8 fibroblasts.

We have shown that 12-O-tetradecanoylphorbol 13-acetate (TPA) increases protein kinase C (PKC)-mediated choline transport, incorporation of choline into phosphatidylcholine (PtdCho) and PtdCho degradation by phospholipase D (PLD) in C3H10T1/2 Cl 8 cells. Dual prelabeling experiment using [3H]/[14C]choline indicated that intracellular choline generated from the PLD reaction was not directly recycled to PtdCho synthesis within the cell, and that a large fraction of the choline was transported out of the TPA-treated cells. In contrast, medium derived choline was preferably channeled to PtdCho synthesis. These results indicate that in TPA-treated cells, the choline derived from the PKC-mediated increased PLD activity and the choline newly taken up by the cell behave as two distinctly different metabolic pools.

[Diagnostic molecular biology in solid tumors--thyroid gland]. / Diagnostisk molekylaerbiologi ved solide svulster--thyreoidea.

Thyroid cancer is not a common disease. It includes tumour types of great diversity in clinical course and molecular basis. Mutations of TSH-receptor, rearrangements of ret proto-oncogene, and altered expression of other tyrosine kinase growth factor receptors are characteristics of the follicular neoplasias and papillary carcinomas, while undifferentiated tumours harbour p53 mutations. Knowledge acquired to date has led to an increased understanding of thyroid growth and tumour development, but it has had no significant impact on diagnostic and treatment measures. On the other hand, the C-cell derived medullary carcinomas include familial cases where identification of germ-line ret mutations provides the basis for prophylactic thyroidectomy in affected individuals.