Publisher Correction: A short isoform of ATG7 fails to lipidate LC3/GABARAP.

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A short isoform of ATG7 fails to lipidate LC3/GABARAP.

Autophagy is a degradation pathway important for cellular homeostasis. The E1-like enzyme ATG7 is a key component of the autophagy machinery, with the main function of mediating the lipidation of LC3/GABARAP during autophagosome formation. By analysing mRNA-sequencing data we found that in addition to the full-length ATG7 isoform, various tissues express a shorter isoform lacking an exon of 27 amino acids in the C-terminal part of the protein, termed ATG7(2). We further show that ATG7(2) does not bind LC3B and fails to mediate the lipidation of members of the LC3/GABARAP family. We have thus identified an isoform of ATG7 that is unable to carry out the best characterized function of the protein during the autophagic response. This short isoform will have to be taken into consideration when further studying the role of ATG7.

Anti-proliferative and pro-apoptotic effects of lichen-derived compound protolichesterinic acid are not mediated by its lipoxygenase-inhibitory activity.

Lipoxygenases (LOXs) and their products are involved in several biological functions and have been associated with carcinogenesis. Protolichesterinic acid (PA), a lichen metabolite, inhibits 5- and 12-LOX and has anti-proliferative effects on various cancer cell lines. Here, PA was shown to inhibit proliferation of multiple myeloma cells, RPMI 8226 and U266, and pancreatic cancer cells AsPC-1. Apoptosis was induced only in multiple myeloma cells. Cell-cycle associated changes in expression and sub-cellular localization of 5- and 12-LOX were not affected by PA but increased cytoplasmic localisation was found to accompany morphological changes at later stages. Assessment by mass spectrometry showed that PA entered the pancreatic cancer cells. However, effects on LOX metabolites were only evident after treatment with concentrations exceeding those having anti-proliferative effects and no effects were measurable in the myeloma cells. We conclude that the anti-proliferative and pro-apoptotic effects of PA are not mediated directly through inhibition of LOX activity.

Longitudinal study of TP53 mutations in eight patients with potentially malignant oral mucosal disorders.

OBJECTIVES: In a previous cross-sectional study, the authors found a higher rate of TP53 mutation in oral lichen planus (OLP) than in hyperkeratosis. By analysing for TP53 mutations in serial samples from patients on long-term follow-up of their oral lesions, it was hoped to determine if these mutations were related to disease progression. METHODS: Eight patients presenting with lesions diagnosed clinically as oral leukoplakia or lichen planus were followed from 2 to 12 years. Two to five samples of archival biopsy tissue were analysed from each patient by constant denaturant gradient gel electrophoresis for hotspots A, B, C, D and exon 6. RESULTS: Four patients were diagnosed clinically as OLP: two of these were confirmed histopathologically, one was diagnosed as non-specific hyperkeratosis and one as cancer. Four patients had leukoplakia only, with a histopathological diagnosis of hyperkeratosis. Seven patients had TP53 mutations, three of them on repeated occasions. All five patients who developed squamous-cell carcinoma had mutations. Two of them had mutated pre-malignant lesions, and one of these previously had a non-mutated cancer. Three patients had two different primary cancers, only one of them mutated. One patient developed a mutated cancer 5 years after the last mutation-free biopsy. Of the cancer-free patients, a lesion regarded clinically as cancer-suspicious in one case was mutated, in another patient two OLP lesions were mutated, the third had five biopsies taken during 8 years, all non-mutated. CONCLUSIONS: TP53 mutations may occur early or late in the development of oral squamous-cell carcinoma.

Role of TP53 in the progression of pre-malignant and malignant oral mucosal lesions. A follow-up study of 144 patients.

BACKGROUND: Prediction of progression from pre-malignant oral mucosal lesions to malignancy, or recurrence of an existing oral squamous cell carcinoma (OSCC), is an important clinical problem in oral medicine. METHODS: This study presents a follow-up of a study published in 2002. Samples from 54 patients with OSCC, 45 with oral lichen planus (OLP) and 45 with hyperkeratosis (clinically leukoplakia), diagnosed between 1987 and 1996, were analysed for TP53 protein expression and TP53 mutation. Follow-up was 11-17 years for OSCC (mean 13.3), 12-22 years for OLP (mean 15.9) and 12-17 years for hyperkeratosis (mean 14.5). RESULTS: Of the 54 OSCC patients, 28 experienced recurrent disease, 21 died of OSCC, 22 died of other causes. Of the 14 OSCC patients with mutated TP53 (n = 11), the cancer recurred in eight (57%) and in 20/39 (51%) without mutation. Expression of TP53 protein was significantly associated with reduced overall survival. Among OLP patients, nine were TP53-mutated out of 31 tested. One TP53-mutated OLP patient developed OSCC in a different site. Of the hyperkeratosis patients, three were mutated of 22 tested. One hyperkeratosis patient (non-mutated) developed OSCC in the same site. CONCLUSION: TP53 mutations can exist in benign oral mucosal lesions for many years without progression to malignancy. No association was found between TP53 protein expression or TP53 mutation and recurrence of OSCC or disease-related survival. Overall survival was reduced in patients with positive TP53 protein expression.

Vbeta usage and T regulatory cells in children following partial or total thymectomy after open heart surgery in infancy.

During open heart surgery in infants the thymus was usually removed, partly or completely. Our previous studies on 16 such children indicated reduced T-cell output later in life with signs of extrathymic maturation of the T cells, but no reduction in T regulatory cells (CD4+CD25+). The diversity of the T-cell repertoire in these children was examined to test if the extrathymic microenvironment could alter Vbeta usage. The expression of Foxp3 and CD127 in CD4+CD25(high) T cells was measured in order to determine whether the T regulatory cells had the phenotype of natural T regulatory cells. There was a wide distribution of Vbeta usage in both study and control groups. Significant variability was found in Vbeta usage for CD4+ and CD8+ T cells when the distribution of the percentage of T cells expressing each Vbeta family was analysed between individuals within each group (P < 0.001; Kruskal-Wallis). Significant difference was also found in average usage of Vbeta2, Vbeta5.1 and Vbeta14 chains within CD4+ T cells and Vbeta2, Vbeta8 and Vbeta21.3 chains within CD8+ cells between the groups (P < 0.05; Student's t-test). There was no difference between the two groups with regard to the proportion of CD4+CD25(high) T cells and no difference in the average expression of Foxp3 or CD127 within the CD4+CD25(high) population. Our data provide evidence that cardiothoracic surgery in infants and total or partial thymectomy alters Vbeta usage, suggesting more limited selection in such children than in the control group. The frequency of natural T regulatory cells seems to be unimpaired.

Evidence for extrathymic T cell maturation after thymectomy in infancy.

Our previous study showed that children who had been partially or completely thymectomized during heart surgery as infants had lower proportions and numbers of total lymphocytes and reduced proportions of T cells (CD3(+)), helper T cells (CD4(+)) and naive T cells (CD3(+) CD4(+) CD45RA(+)), but normal proportion of cytotoxic T cells (CD8(+)). In this study T lymphocytes from a selected group of eight of these children and age- and gender-matched controls were characterized further using flow cytometry to determine phenotypes of T cells and T cell subsets related to T cell regulation and phenotypes suggestive of extrathymic maturation. Immune function was assessed by measuring autoantibodies and antibodies against vaccines. The study group had significantly lower numbers of all the main subsets of T lymphocytes and the composition was different. Thus, the proportions of lymphocytes with the following phenotypes: CD3(+), CD2(+), CD7(+), CD4(+), CD62L(+), CD4(+) CD62L(+) and CD4(+) CD69(-) were significantly reduced in the study group compared with the control group, but significantly higher proportions were seen of lymphocytes expressing CD8alpha(+) CD8beta(-) and TCRgammadelta(+) CD8alpha(+) CD8beta(-). The absolute number and proportion of CD4(+) CD25(+) cells were reduced but the proportions of the subgroup of naive regulatory T cells (CD4(+) CD25(+) CD62L(+)) and non-activated regulatory T cells (CD4(+) CD25(+) CD69(-)) were not reduced in the thymectomized children. We conclude that the phenotypic characteristics of T lymphocytes of children who have lost their thymus in infancy are indicative of extrathymic maturation. T regulatory cells appear to be less affected than other subsets by the general reduction in T cell numbers.

Positive association between plasma antioxidant capacity and n-3 PUFA in red blood cells from women.

PUFA are susceptible to oxidation. However, the chain-reaction of lipid peroxidation can be interrupted by antioxidants. Whether an increased concentration of PUFA in the body leads to decreased antioxidant capacity and/or increased consumption of antioxidants is not known. To elucidate the relationship between plasma total antioxidant capacity (TAC), the concentration of antioxidant vitamins, and the proportion of PUFA in red blood cells (RBC), plasma TAC was measured by a Trolox equivalent antioxidant capacity assay in blood samples from 99 Icelandic women. Concentrations of tocopherols and carotenoids in the plasma were determined by HPLC, and the FA composition of RBC total lipids was analyzed by GC. Plasma TAC and the plasma concentration of alpha-tocopherol correlated positively with the proportion of total n-3 PUFA, 20:5n-3, and 22:6n-3 in RBC, whereas the plasma lycopene concentration correlated negatively with the proportion of total n-3 PUFA and 20:5n-3. On the other hand, plasma TAC correlated negatively with the proportion of n-6 PUFA in RBC. Plasma TAC also correlated positively with the plasma concentration of alpha-tocopherol, alcohol consumption, and age. Both the plasma concentration of alpha-tocopherol and age correlated positively with the proportion of n-3 PUFA in RBC; however, n-3 PUFA contributed independently to the correlation with plasma TAC. Because the proportion of n-3 PUFA in RBC reflects the consumption of n-3 PUFA, these results suggest that dietary n-3 PUFA do not have adverse effects on plasma TAC or the plasma concentration of most antioxidant vitamins.

The influence of partial or total thymectomy during open heart surgery in infants on the immune function later in life.

Infants undergoing open heart surgery often have all or part of their thymus removed. The activity of the immune system has not been investigated thoroughly in these children, and only shortly after the operation. Therefore, it was decided to investigate the activity of the immune system in more detail in children several years after their operation. Peripheral blood samples from 19 children who had undergone open heart surgery during their first months of life was collected (study group) and from 19 age- and gender-matched children (control group). The activity of the immune system was evaluated by measuring the number of different cell types in peripheral blood, the phenotype of lymphocytes and the response of T cells following in vitro stimulation by mitogen, tetanus toxoid and measles antigen. The study group had significantly lower counts of total lymphocytes, which was reflected in a lower number of T cells but not B cells. Furthermore, the study group had significantly lower proportion of T cells (CD3(+)) and helper T cells (CD4(+)), but not cytotoxic T cells (CD8(+)). The level of neutrophils in peripheral blood was significantly higher in the study group. This may indicate enhanced innate immunity when the acquired immunity is defective. The results indicate a shift to extrathymic T cell maturation, which is less efficient for CD4(+) helper cells than for CD8(+) cytotoxic cells.

Effects of tenuiorin and methyl orsellinate from the lichen Peltigera leucophlebia on 5-/15-lipoxygenases and proliferation of malignant cell lines in vitro.

The orcinol derivatives tenuiorin (1) and methyl orsellinate (2) were identified as active components of an extract from the lichen Peltigera leucophlebia (Nyl.) Gyeln. showing in vitro inhibitory activity against 15-lipoxygenase from soybeans. The compounds were subsequently tested for in vitro activity against 5-lipoxygenase from porcine leucocytes and proved to be moderately active, with IC50 values of 41.6 microM and 59.6 microM respectively. Tenuiorin is a known constituent of several Peltigera species but has not previously been isolated from P. leucophlebia. As correlation between 5-lipoxygenase inhibition and antiproliferative effects has earlier been witnessed for related lichen metabolites, tenuiorin and methyl orsellinate were further tested for antiproliferative activity on cultured human breast (T-47D)-, pancreatic (PANC-1)- and colon (WIDR) cancer cell lines. The monomeric methyl orsellinate exhibited no detectable antiproliferative activity whereas the trimeric tenuiorin caused moderate/weak reduction in [3H]-thymidine uptake of the pancreatic- and colon cancer cells, with ED50 values of 87.9 and 98.3 microM respectively.

The effect of a single BRCA2 mutation on cancer in Iceland.

OBJECTIVE: To estimate the risk of malignant diseases in families of probands with the same mutation in the BRCA2 gene. DESIGN: A cohort study using record linkage of a breast cancer family resource and the Icelandic Cancer Registry. SETTING: Iceland. SUBJECTS: Families of 995 breast cancer patients, from which 887 were tested for a single founder 999del5 mutation; 90 had the mutation and 797 did not. RESULTS: Relatives of probands with the mutation had significantly increased relative risk (RR) of breast cancer. For first degree relatives, the RR was 7.55 (95% CI 6.04 to 9.03) but was 1.72 (95% CI 1.49 to 1.96) in first degree relatives of probands without the mutation. For prostate and ovarian cancer, the first and second degree relatives of probands with the mutation had a significantly increased RR, but in families of probands without the mutation no significant familial risk was found. CONCLUSIONS: The 999del5 mutation in the BRCA2 gene explains a substantial proportion of familial risk of breast cancer in Iceland, but significant familial risk remains in relatives of probands without the mutation. For prostate and ovarian cancer, the mutation accounts for most of the familiality observed in families of breast cancer patients.

Immune reaction to breast cancer: for better or for worse?

The infiltration of breast carcinomas with lymphoid cells has often been interpreted as an indication of an active immune response against the tumour and, thus, a favourable prognostic sign. Several studies have, however, cast doubt on this assumption. In situ breast carcinomas are more common than invasive cancers, and it may be speculated that immune surveillance plays a role in preventing some localized cancers from becoming invasive. A secondary type of immune surveillance might be implicated in the long persistence of dormant breast carcinoma cells in the bone marrow. Breast cancer cells can carry tumor-associated antigens, particularly MUC1. These may elicit specific antibody responses, but there is less evidence for a cytotoxic T lymphocyte (CTL) response. There are indications that professional antigen-presenting cells (APC) may be present and active at the edges of breast tumours. Breast cancer cells may also interact directly with macrophages and natural killer (NK) cells. In terms of immune effector mechanisms in breast cancer, the communication with potential effector cells is likely to be often faulty because of altered expression of HLA class I molecules. Pleiotrophic cytokines are frequently present and could have a variety of effects ranging from growth inhibition to stimulated proliferation, loss of cell adhesion and activation of matrix-degrading enzymes. Fas ligand is unlikely to play a role in the immune evasion of breast cancer. There is thus evidence for a variety of immune reactions to breast cancer. It is possible that they mediate some form of surveillance, but growing, invasive tumours have escape routes and may even use cytokines to their advantage.

Helicobacter pylori antibodies and gastric cancer in Iceland - The decline in IgG antibody level is a risk factor.

H. pylori infection is considered a causal agent of duodenal ulcer and a significant risk factor for gastric cancer. Retrospective cohort studies have demonstrated a significant association between presence of antibody to H. pylori and gastric cancer when using samples obtained years before the diagnosis but not at the time of diagnosis. The present study investigates, in a population-based cohort, whether a decline occurs in H. pylori antibody levels before the diagnosis of stomach cancer. Repeat samples (2 to 5) were available from 23 persons with gastric cancer taken up to 20 years before the diagnosis and 128 control subjects matched for gender, age, time and number of repeat samples. The odds ratio of developing stomach cancer was 1.16 (95% CI 1.05-1.28) for those showing decline in antibody levels of 1 relative antibody activity unit per year versus those with constant or rising levels. We conclude that this decline in antibody levels in cases, and not in controls, supports an active role of H. pylori in the pathogenesis of gastric cancer by causing atrophic gastritis, and provides a better risk assessment for gastric cancer compared to single measurements.

Intracellular Fas ligand in normal and malignant breast epithelium does not induce apoptosis in Fas-sensitive cells.

Fas ligand (FasL) is expressed on some cancers and may play a role in the immune evasion of the tumour. We used immuno-histochemistry to study the expression of Fas and FasL in tissue samples from breast cancer patients, as well as normal breast tissue. Our results show that Fas and FasL are co-expressed both in normal tissue and in breast tumours. Fas and FasL mRNA were expressed in fresh normal and malignant breast tissue, as well as cultured breast epithelium and breast cancer cell lines. Flow cytometry analysis of live cells failed to detect FasL on the surface of normal or malignant breast cells; however, both stained positive for FasL after permeabilization. Fas was detected on the surface of normal breast cells and T47D and MCF-10A cell lines but only intracellularly in other breast cell lines tested. Neither normal breast epithelium nor breast cell lines induced Fas-dependent apoptosis in Jurkat cells. Finally, 20 tumour samples were stained for apoptosis. Few apoptotic cells were detected and there was no increase in apoptotic cells on the borders between tumour cells and lymphocytes. We conclude that FasL is expressed intracellularly in both normal and malignant breast epithelium and unlikely to be important for the immune evasion of breast tumours.

Altered expression of HLA class I antigens in breast cancer: association with prognosis.

Loss of surface expression of class I major histocompatibility antigens is commonly observed in malignant tumors and has been considered one of the mechanisms for escape from cytotoxic T cells. However, natural killer cells kill cells lacking HLA class I antigens. In the present study, we characterized by immunohistochemistry the HLA class I expression of breast carcinomas from 187 patients with TNM stages I and II, diagnosed 1981-1984, using beta(2)-microglobulin as a marker and evaluated the effect on survival with a follow-up of up to 14 years. The largest group (48%) consisted of HLA class I-negative tumors (< or =10% of cells stained), mixed expression (>10% and <80% of cells stained) was seen in 36% and only 15% were classified as HLA class I-positive (> or=80% cells stained). No associations could be established with various clinicopathological parameters, such as tumor size, presence of lymph node metastases, histological grade, expression of hormone receptors, S phase and p53 mutations. There was no effect on recurrence-free survival in the whole group; but among node-negative patients (n = 86), those who had tumors with mixed HLA class I expression had a significantly higher probability of disease recurrence (OR = 3.42, p = 0.014) than patients with either HLA class I-positive or -negative tumors, particularly after more than 5 years. In node-positive patients who received adjuvant therapy, this phenotype was not associated with disease recurrence.

p53 abnormality and chromosomal instability in the same breast tumor cells.

To clarify the important role of the tumor-suppressor gene p53 in maintaining genetic integrity, we estimated chromosome instability and staining of overexpressed p53 protein in the same cells of five primary breast carcinomas. The method included both fluorescence immunohistochemistry and fluorescence in situ hybridization (FISH) on sections from formalin-fixed, paraffin-embedded breast cancer tissue. By using a centromeric FISH probe for chromosome 17 on interphase cells in these sections, we showed that cells with abnormal p53 protein expression had a statistically significant higher number of chromosome 17 than did cells with no p53 protein staining in the same samples as well as cells in four other tumor samples with no p53 protein staining. The samples identified positive for p53 abnormality by immunostaining were shown to have p53 mutation by constant denaturing gel electrophoresis analysis and DNA sequencing. These mutated samples were characterized by high DNA index, high S-phase, abnormal karyotype, and aneuploidy. The results strongly implicate p53 mutation as a cause for chromosomal instability and a crucial step in mammary carcinogenesis.

Altered expression of E-cadherin in breast cancer. patterns, mechanisms and clinical significance.

Reduced cell adhesion brought about by altered surface expression of E-cadherin has been implicated in invasive and metastatic malignant growth. We investigated the patterns of immunohistochemical E-cadherin expression in 120 breast carcinomas. Furthermore, we analysed DNA from the same samples for loss of heterozygosity (LOH) using three separate microsatellite markers on chromosome 16q22.1. Finally, the clinical outcome was ascertained for 108 patients. 19% (18/97) of infiltrating ductal carcinomas showed complete loss of E-cadherin expression compared with 64% (9/14) of infiltrating lobular carcinomas. LOH was detected in 46% (24/52) of infiltrating ductal carcinomas and 89% (8/9) of infiltrating lobular carcinomas. In the infiltrating lobular carcinomas, LOH was associated with complete loss of cell membrane expression of E-cadherin, although a cytoplasmic expression pattern was evident. In contrast, this association was not seen in the infiltrating ductal carcinomas. In a multivariate analysis, loss of E-cadherin expression was shown to be a significant independent risk factor for a poorer disease-free survival (P=0.019), in particular in the node-negative subset of patients (P=0.029). Significance was also approached for breast cancer corrected survival (P=0.056). We conclude that different mechanisms are involved in the altered E-cadherin expression seen in different subtypes of breast carcinomas. Furthermore, we implicate loss of E-cadherin, regardless of the genetic causes, as an independent prognostic marker for disease recurrence, especially in node-negative breast cancer patients, irrespective of the histological type.

Enhanced B cell survival in familial macroglobulinaemia is associated with increased expression of Bcl-2.

A family with three cases of macroglobulinaemia of undetermined significance (MGUS), and one case each of immunoblastic lymphoma, Waldentröm's macroglobulinaemia and multiple myeloma was first described 20 years ago. We have previously identified 10 out of 35 healthy family members tested whose lymphocytes produced abnormally high amounts of immunoglobulins in culture. In the present study lymphocyte subpopulations of these hyper-responders have been further characterized and lymphocyte reactivity and survival in vitro have been studied. No differences were detected in the proportions of resting B lymphocytes (CD19+) co-expressing CD5, CD10, CD11b, or CD38, and the CD4/CD8 ratio of T cells was normal before and after stimulation with pokeweed mitogen (PWM). The initial rate of response in terms of immunoglobulin production was not increased, but immunoglobulin levels continued to rise during the second week of culture whereas the production peaked at 8 days in control cultures. This was associated with significantly greater survival of lymphocytes and at 14 days surviving B cells could only be identified in samples from hyper-responders. A lymph node removed because of tuberculosis from a family member 23 years before the diagnosis of multiple myeloma showed very marked Bcl-2 expression in a B cell follicle. This was not seen in a tuberculous lymph node from an unrelated subject. Stimulated cultures from three hyper-responders tested demonstrated significantly higher retention of Bcl-2 in B cells compared with one family control and six unrelated controls. We conclude that the increased production of immunoglobulins previously observed in this family with an inherited tendency for benign and malignant B cell proliferation is the result of enhanced B cell survival, which is associated with increased expression of Bcl-2 following stimulation.

The effects of IL-6 on cell adhesion and e-cadherin expression in breast cancer.

Interleukin 6 (IL-6) is a pleiotropic inflammatory cytokine and its role in cancer is not yet clear. The effects of IL-6 on four breast cancer cell lines and normal mammary epithelium, cultured from milk were tested. Four different patterns of response to IL-6 were found depending on the differentiation status of the cells. In normal mammary epithelial cultures, the effects of IL-6 were mainly growth inhibitory, whereas in MCF-7, IL-6 had growth inhibitory and anti-adhesive effects. In T-47D and ZR-75-1 the anti-adhesive effects were prominent although the growth inhibitory effects were not. These anti-adhesive effects were associated with epithelioid to fibroblastoid morphological changes and a local decrease in E-cadherin expression. In the highly invasive cell line MDA-MB-231, which does not express E-cadherin, no effects of IL-6 were seen. IL-6 levels in the serum of 60 breast cancer patients were found to be increased in 27% (16/60) compared to 2% (1/50) in a control group. Furthermore, it was found that altered E-cadherin expression was seen in 69% of the primary tumours, although no significant association was found between raised serum IL-6 levels and altered E-cadherin expression. Finally IL-6 serum levels did not effect the survival of breast cancer patients. The authors therefore implicate IL-6 as a possible factor important in breast cancer progression and metastasis formation, although the clinical significance of this cytokine in breast cancer patients could not be established.