Biomedical subjects

H Blegen

Publications and source records attributed to H Blegen.

At least 19 recordsLinked to original sources

PubMed2001

Frequent co-amplification of two different regions on 17q in aneuploid breast carcinomas.

Chromosome 17q is highly susceptible to rearrangement mutations in breast cancer. c-erbB-2 at 17q11.2 approximately q21.1 is frequently amplified, as is a region at 17q22 approximately q24. As a step in the search for the target gene(s) of the 17q22-q24 amplification we determined whether the placental lactogen (PL) genes at 17q23 were amplified in 59 breast carcinomas. These genes were selected as their upregulation could theoretically be involved in breast cancer tumorigenesis. Amplification of the PL genes, and also of c-erbB-2, was detected using semi-quantitative PCR. The reliability of this method was confirmed since c-erbB-2 results obtained using PCR, Southern blotting and immunohistochemistry were in good agreement. The PL genes were amplified in 13 (22%) of the tumors. Furthermore, the PL and c-erbB-2 genes were frequently co-amplified although there is a non-amplified region between them. Expression of PL was investigated in 26 tumors and was detected in 16 of these cases including all 10 tumors with amplification of the PL genes. The tumors with PL gene amplification were all aneuploid. A trend was seen towards an increased incidence of lymph node involvement for tumors with amplification of the PL genes and for tumors with co-amplification of PL and c-erbB-2, which suggests a possible association with high malignancy.

Aneuploidy↗

PubMed2001

Genetic instability promotes the acquisition of chromosomal imbalances in T1b and T1c breast adenocarcinomas.

In order to evaluate biological and genetic properties of early breast carcinomas we analyzed microdissected tissue from 33 primary breast carcinomas stage T1b and T1c with respect to the nuclear DNA content, the expression pattern of Ki-67, cyclin A, p27KIP1, p53 and p21WAF1, and chromosomal gains and losses. The results show that T1b carcinomas (6-10 mm, n=17) were frequently near-diploid (53%) with low proliferative activity and staining patterns of p53 and p21WAF1 that suggest the presence of wild type protein. The majority (12/16) of the T1c tumors (11-20 mm), however, was aneuploid, and proliferative activity and p53 expression were increased. Larger tumor size correlated with an increasing number of chromosomal copy number changes and in particular with regional amplifications. High level copy number increases (amplifications), however, were found exclusively in the aneuploid tumors. Amplification events correlated with elevated cyclin A and reduced p27 expression, respectively. Our results suggest that the sequential acquisition of genomic imbalances during tumor progression is accelerated in aneuploid tumors, and may contribute to the increased malignancy potential.

Adenocarcinoma↗

PubMed2000

Recurrent fallopian tube carcinoma: TP53 mutation and clinical course.

Primary fallopian tube carcinoma is a rare, aggressive gynecological cancer; little is known about its cause. Previous studies have indicated that p53 immunopositivity is correlated with short-term survival in primary fallopian tube carcinoma. We examined p53 and p21/WAF1 immunostaining and TP53 mutation in exons 5 to 8 by single-stranded conformation polymorphism and constant denaturant gel electrophoresis in nine cases of primary fallopian tube carcinoma and their metastases/recurrences from patients who survived for between a few months and more than 20 years after diagnosis. We found that 1.) p53 immunopositivity without detectable p21/WAF1 immunostaining did not correlate with TP53 mutations in the conserved domains; 2.) mutations in TP53 occurred in two metastases/recurrences but not in their corresponding primary tumors; 3.) in two cancers, a TP53 mutation was observed in the primary tumor but not in the metastases/recurrences; 4.) constant denaturant gel electrophoresis seems to be more sensitive than single-stranded conformation polymorphism in detecting TP53 mutations; and 5.) in the nine cases studied, p53 immunoreactivity and/or TP53 mutation analysis did not correlate with tumor progression, survival, or response to treatment.

Aged↗

PubMed1999

Genomic changes defining the genesis, progression, and malignancy potential in solid human tumors: a phenotype/genotype correlation.

The transition of normal epithelium to invasive carcinoma occurs sequentially. In colorectal and cervical carcinogenesis, this transition is reflected by histomorphologically defined grades of increasing dysplasia that untreated may progress to invasive disease. In an attempt to understand the role of chromosomal aberrations during tumorigenesis we have applied comparative genomic hybridization using DNA extracted from defined stages of colorectal and cervical tumors, from low- and high-grade astrocytic tumors and from diploid and aneuploid breast carcinomas. Genetic instability, as measured by the number of chromosomal copy alterations per case, increases significantly at the transition from precursor lesions to invasive carcinomas and continues to increase with tumor stage. Aggressive tumors have a higher number of copy alterations per case. High-level copy number changes (amplifications) become more prevalent in advanced-stage disease. Subtractive karyograms of chromosomal gains and losses were used to map tumor stage-specific chromosomal aberrations and clearly showed that nonrandom chromosomal aberrations occur during disease progression. In colorectal and cervical tumors, chromosomal copy number changes were correlated with nuclear DNA content, proliferative activity, expression levels of the tumor suppressor gene TP53, and the cyclin-dependent kinase inhibitor p21/WAF1, as well as the presence of viral genomes. Here we summarize and review the results of this comprehensive phenotype/genotype correlation and discuss the relevance of stage-specific chromosomal aberrations with respect to diagnostic applications.

Disease Progression↗

PubMed1998

Primary carcinoma of the fallopian tube: comparative genomic hybridization reveals high genetic instability and a specific, recurring pattern of chromosomal aberrations.

Primary fallopian tube carcinoma (PFTC) is a rare and highly aggressive tumor. Twelve cases of PFTC (stages IA to IV) were analyzed by comparative genomic hybridization. The most consistent DNA gain was mapped to chromosome arm 3q in 11 of 12 cases. In six cases, the gain of 3q was present as a high level copy number increase (amplification) with a consensus region mapped to 3q26.2-qter. In the 12 cases, other frequent gains were located on chromosome arms 1q (in 11 cases), 2q (in 10), 7q (in 9), 8q (in 9), 5p (in 8), 6p (in 7), 12p (in 7), and 14q (in 6). Frequent copy number losses occurred on chromosome arms 16q (in 8 cases), 22q (in7), 6q (in 6). 8p (in 6), 18q (in 6), Xq (in 6), 1p (in 5), and 17p (in 5). All chromosomes were involved in chromosomal aberrations and the average number of copy alterations per case was 19.7. None of the 12 carcinomas revealed the presence of human papillomavirus (HPV) genomes. All of the cases exhibited crude aneuploidy. Strong p53 immunoreactivity could be observed in 10 of 12 cases while p21/WAF1 expression was low or undetectable. These results indicate that PFTC is a genomically highly unstable cancer, an observation that is in agreement with the poor prognosis associated with this tumor. A high frequency of 3q-gains has also been observed in HPV-related carcinomas of the uterine cervix. However, none of the PFTC was HPV related, suggesting that the 3q-gain is independent from HPV DNA.

Aged↗

PubMed1997

A recurrent pattern of chromosomal aberrations and immunophenotypic appearance defines anal squamous cell carcinomas.

Squamous cell carcinomas of the anus are rare neoplasias that account for about 3% of large bowel tumours. Infections with human papillomaviruses are frequently detected in these cancers, suggesting that pathogenic pathways in anal carcinomas and in carcinomas of the uterine cervix are similar. Little is known regarding recurrent chromosomal aberrations in this subgroup of squamous cell carcinomas. We have applied comparative genomic hybridization to identify chromosomal gains and losses in 23 cases of anal carcinomas. A non-random copy number increase of chromosomes 17 and 19, and chromosome arm 3q was observed. Consistent losses were mapped to chromosome arms 4p, 11q, 13q and 18q. A majority of the tumours were aneuploid, and most of them showed increased proliferative activity as determined by staining for Ki-67 antigen. p53 expression was low or undetectable, and expression of p21/WAF-1 was increased in most tumours. Sixteen cancers were satisfactorily tested for the presence of HPV by consensus L1-primer polymerase chain reaction; nine were HPV positive, of which eight were positive for HPV 16.

Adult↗

PubMed1997

Mevalonate-regulated mechanisms in cell growth control: role of dolichyl phosphate in expression of the insulin-like growth factor-1 receptor (IGF-1R) in comparison to Ras prenylation and expression of c-myc.

One or more mevalonate derivatives of non-sterol type have been proposed to be of indispensable importance for cell growth. Conceivable mevalonate-dependent mechanisms involved in growth control are farnesylation of Ras proteins, regulation of c-myc expression, and N-linked glycosylation of the IGF-1 receptor. The latter mechanism might be rate-limited by dolichyl phosphate, which acts as a donor of oligosaccharides in glycoprotein synthesis in the endoplasmic reticulum. In order to study the significance for cell proliferation of the three aforementioned mevalonate-dependent processings, their inhibitory response due to mevalonate deprivation was explored and compared with the effect on DNA synthesis in the malignant melanoma cell line SK-MEL-2. We found that mevalonate depletion due to treatment with 3 microM lovastatin for 24 h, which efficiently growth-arrested the cells, hardly at all affected the expression of c-myc, and although Ras prenylation was inhibited by 50%, the most pronounced effect of lovastatin was seen on N-linked glycosylation of IGF-1 receptors, which was inhibited by more than 95%. The order and magnitude of the decreased IGF-1 receptor glycosylation, which was followed by a decreased expression of IGF-1 receptors at the cell membrane, correlated well with the inhibition of DNA synthesis. We investigated whether dolichol, and in particular dolichyl phosphate, through its participation in N-linked glycosylation, act as regulators of IGF-1 receptor expression. First, we could confirm that exogenous dolichol became phosphorylated and in this form took part in the glycosylation processing. Secondly, we showed that dolichyl phosphate, in a dose-dependent manner, could increase the number of IGF-1 receptors at the cell membrane, simultaneously as DNA synthesis was stimulated. Taken together, our results provide direct evidence for an important role of dolichyl phosphate as a regulator of cell growth through limiting N-linked glycosylation of the IGF-1 receptor.

Cell Division↗

PubMed1997

Advanced-stage cervical carcinomas are defined by a recurrent pattern of chromosomal aberrations revealing high genetic instability and a consistent gain of chromosome arm 3q.

We have analyzed 30 cases of advanced-stage cervical squamous cell carcinoma (stages IIb-IV) by comparative genomic hybridization (CGH). The most consistent chromosomal gain in the aneuploid tumors was mapped to chromosome arm 3q in 77% of the cases. Acquisition of genetic material also occurred frequently on Iq (47%), 5p (30%), 6p (27%), and 20 (23%). Recurrent losses were mapped on 2q (33%), 3p (50%), 4 (33%), 8p (23%), and 13q (27%). High-level copy number increases were mapped to chromosome 8, chromosome arms 3q, 5p, 8q, 12p, 14q, 17q, 19q, 20p, and 20q, and chromosomal bands 3q26-27, 9p23-24, 11q22-23, and 12p13. In the majority of the cases, the presence of high-risk human papilloma virus genomes was detected. High proliferative activity was accompanied by crude aneuploidy. Increased p21/WAF-I activity, but low or undetectable expression of TP53 were representative for the immunophenotype. This study confirms the importance of a gain of chromosome arm 3q in cervical carcinogenesis and identifies additional, recurrent chromosomal aberrations that are required for progression from stage I tumors to advanced-stage carcinomas.

Adult↗

PubMed1996

Mevalonic acid is limiting for N-linked glycosylation and translocation of the insulin-like growth factor-1 receptor to the cell surface. Evidence for a new link between 3-hydroxy-3-methylglutaryl-coenzyme a reductase and cell growth.

Depletion of mevalonic acid (MVA), obtained by inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase using lovastatin, depressed the biosynthesis of dolichyl-phosphate and the rate of N-linked glycosylation and caused growth arrest in the melanoma cell line SK-MEL-2. The growth arrest was partially prevented by addition of high concentrations of insulin-like growth factor-1 (IGF-1) to the cells, indicating that MVA depletion may inhibit cell growth through decreasing the number of IGF-1 receptors (IGF-1R) at the cell surface. Such a decrease in receptor number might be a result of a lowered translocation of de novo synthesized receptors to the cell membrane which in turn might be a result of a decreased N-linked glycosylation of the receptor proteins. We could also demonstrate that IGF-1R became underglycosylated and that the amount of de novo synthesized IGF-1R proteins at the cell membrane was drastically decreased upon MVA depletion. Analysis of receptor proteins cross-linked with IGF-1, as well as binding assays and immunocytostaining confirmed that the number of functional membrane-bound IGF-1R was substantially reduced. The N-linked glycosylation and the expression of de novo synthesized IGF-1R proteins at the cell surface as well as the number of IGF-1 binding sites were completely restored upon replenishment of MVA. These effects of MVA were efficiently abrogated by the glycosylation inhibitor tunicamycin. The translocation of IGF-1R to the cell membrane was shown to take place just prior to initiation of DNA synthesis in arrested cells stimulated with MVA. Additionally, there was a clear correlation between IGF-1 binding and initiation of DNA synthesis with regard to the MVA dose requirement. It was confirmed that inhibition of HMG-CoA reductase activity and N-linked glycosylation also depressed the expression of functional IGF-1R in other cell types (i.e. hepatoblastoma cells and colon cancer cells). Our data suggest that this mechanism is involved in MVA-regulated cell growth.

Base Sequence↗

PubMed1996

Gain of chromosome 3q defines the transition from severe dysplasia to invasive carcinoma of the uterine cervix.

We have chosen tumors of the uterine cervix as a model system to identify chromosomal aberrations that occur during carcinogenesis. A phenotype/genotype correlation was established in defined regions of archived, formalin-fixed, and hematoxylin/eosin-stained tissue sections that were dissected from normal cervical epithelium (n = 3), from mild (n = 4), moderate (n = 6), and severe dysplasias/carcinomas in situ (CIS) (n = 13), and from invasive carcinomas (n = 10) and investigated by comparative genomic hybridization. The same tissues were analyzed for DNA ploidy, proliferative activity, and the presence of human papillomavirus (HPV) sequences. The results show that an increase in proliferative activity and tetraploidization had occurred already in mildly dysplastic lesions. No recurrent chromosomal aberrations were observed in DNA extracted from normal epithelium or from mild and moderate dysplasias, indicating that the tetraploidization precedes the loss or gain of specific chromosomes. A gain of chromosome 3q became visible in one of the severe dysplasias/CIS. Notably, chromosome 3q was overrepresented in 90% of the carcinomas and was also found to have undergone a high-level copy-number increase (amplification). We therefore conclude that the gain of chromosome 3q that occurs in HPV16-infected, aneuploid cells represents a pivotal genetic aberration at the transition from severe dysplasia/CIS to invasive cervical carcinoma.

Aneuploidy↗

PubMed1996

Comparative genomic hybridization reveals a specific pattern of chromosomal gains and losses during the genesis of colorectal tumors.

Comparative genomic hybridization was used to screen the DNA extracted from histologically defined tissue sections from consecutive stages of colorectal carcinogenesis for chromosomal aberrations. No aberrations were detected in normal epithelium (n = 14). Gain of chromosome 7 occurred as a single event in low-grade adenomas (n = 14). In high-grade adenomas (n = 12), and overrepresentation of chromosomes 7 and 20 was present in 30% of the cases analyzed. The transition to colon carcinomas (n = 16) was characterized by the emergence of multiple chromosomal aberrations. Chromosomes 1, 13, and 20 and chromosome arms 7p and 8q were frequently gained, whereas chromosome 4 and chromosome arms 8p and 18q were recurrently underrepresented. The same tissue sections that were used for CGH were analyzed by means of DNA-ploidy measurements and immunohistochemical staining to quantify proliferative activity and p21/WAF-1 and TP53 expression. We observed that crude aneuploidy and increased proliferative activity are early events in colorectal carcinogenesis, followed by TP53 overexpression and the acquisition of recurrent chromosomal gains and losses during the progression from high-grade adenomas to invasive carcinomas.

Adult↗

PubMed1996

Short exposures to tunicamycin induce apoptosis in SV40-transformed but not in normal human fibroblasts.

When SV40-transformed fibroblasts (line 90VAVI) were exposed to tunicamycin, an inhibitor of N-linked glycosylation, an extensive cell death occured compared with untransformed fibroblasts. A considerable cell loss was obtained within 24 h after tunicamycin addition, and after 72 h there were hardly any virus-transformed cells alive. A 2-h pulse treatment with tunicamycin was found to be almost as effective as a continuous 48-h treatment in killing the cells. Even such a short exposure as 7 min resulted in a drastically decreased cell viability (54%). The morphology of the dying tunicamycin-treated 90VAVI cells suggested that they were undergoing apoptosis. This was also supported by the appearance of nuclear condensation, as assayed by propidium iodide uptake, which was detectable within 2 h after tunicamycin addition. Furthermore, analysis of DNA from tunicamycin-treated 90VAVI cells by field inversion gel electrophoresis revealed DNA degradation into 50 kbp fragments within 2 h, and conventional agarose gel electrophoresis showed 'DNA laddering', indicating internucleosomal DNA cleavage, detectable after 36 h. Together with the finding that tunicamycin within seconds caused an elevation of [Ca2+]i, a well documented early feature of apoptosis in many experimental systems, these results strongly suggest that tunicamycin-induced cell death in 90VAVI is due to apoptosis. The short tunicamycin exposure required to trigger cell death in 90VAVI indicates that the apoptotic process is irreversibly induced soon after its addition. It seems unlikely that the pool of one or several specific N-linked glycoproteins could be depleted during such a short period. Instead the overall accumulation of unglycosylated proteins in ER might contribute to the apoptotic response in 90VAVI. Tunicamycin also killed and induced DNA degradation in the breast cancer cell line MDA-231.

Apoptosis↗

PubMed1995

Comparative genomic hybridization of formalin-fixed, paraffin-embedded breast tumors reveals different patterns of chromosomal gains and losses in fibroadenomas and diploid and aneuploid carcinomas.

Comparative genomic hybridization serves as a screening test for regions of copy number changes in tumor genomes. We have applied the technique to map DNA gains and losses in 33 cases of formalin-fixed, paraffin-embedded primary breast tumors (13 fibroadenomas and 10 diploid and 10 aneuploid carcinomas). No genomic imbalances were found in fibroadenomas. Recurrent findings in adenocarcinomas include copy number increases for chromosomes 1q (14 of 20 samples), 8q (10 of 20), 17q (5 of 20), 6p (3 of 20), 13q (3 of 20), and 16p (3 of 20), and copy number decreases for chromosomes 22 (7 of 20), 17p (6 of 20), and 20 (3 of 20). Regional high level copy number increases were observed on chromosome bands 1q32, 8p11, 8q24, 10p, 11q13, 12p, 12q15, 17q11-12, and 17q22-24. The majority of the samples were studied for gene amplification of c-myc, c-erbB2, cycD1, and int-2 by means of Southern blot analysis. The comparison with DNA ploidy measurements revealed a different distribution and a significantly higher number of chromosomal aberrations in aneuploid tumors than in diploid tumors and in fibroadenomas.

Aneuploidy↗

PubMed1995

Utility of a proliferation marker in distinguishing between benign naevocellular naevi and naevocellular naevus-like lesions with malignant properties.

In the present study we have investigated the utility of the proliferation marker MIB 1 in distinguishing between benign naevocellular naevi and naevocellular naevus-like lesions with malignant potential. Percentages of MIB 1 immunoreactivity in the intradermal portion of the lesions were determined. In benign congenital and acquired naevi, as well as in dysplastic naevi, there was no or only a slight intradermal melanocytic proliferation (0-2%), whereas vertical growth phase melanomas exhibited a substantial proliferative activity (11-48%). In five cases of naevus-lke lesions, which had all relapsed as unmistakable malignant melanomas (locally or metastatically) after primary surgery, there was also clear proliferative activity (9-67%). Our findings suggest that MIB 1 may be a useful tool in the routine histopathological examination of problematic naevocellular lesions.

Antibodies, Monoclonal↗

PubMed1994

Inhibitory effect of mevalonate on the EGF mitogenic signaling pathway in human breast cancer cells in culture.

The relationship between the effects of EGF and mevalonate on proliferation of the breast cancer cell line Hs578T was investigated. When Hs578T cells were depleted of serum their proliferation was drastically retarded. This was partially counteracted by insulin or IGF-1 but not by EGF. However, if the activity of HMG-CoA reductase was inhibited, there was a significant increase in DNA synthesis of EGF-treated cells. This effect was not seen in cells stimulated by insulin or IGF-1, and was prevented by addition of mevalonate. The results suggest that mevalonate, or some of its products, inhibits steps in the EGF signal pathway.

Breast Neoplasms↗

PubMed1993

A cell cycle study of human mammary epithelial cells.

The growth regulation of human mammary epithelial cells (HMEC) cultured in a growth factor/hormone-enriched (e.g. EGF, insulin) medium with bovine pituitary extract as the only undefined supplement was studied. The doubling times of the cultures, in which the cells appear in colonies, was 55-72 h, and a considerable intercolonial heterogenecity in proliferative activity could be demonstrated. However, every colony, irrespective of the size of the growth fraction, comprised a sub-population of rapidly growing cells which had a mean generation time of approximately 22 h. When insulin was removed from the culture medium, HMEC proliferation was inhibited. This growth inhibition was shown to be a result of a cell cycle-specific block.

Adult↗

PubMedDate not supplied

A rat model for severe limb ischemia at rest.

We sought an animal model able to discriminate metabolic and angiogenic processes in limb ischemia. For that we modified and evaluated a rat model of severe unilateral limb ischemia at rest. A two-stage surgical procedure entailing left femoral artery ligation preceded by interruption of collateral vessels originating from the infra-renal aorta and left iliac arteries was performed in Sprague-Dawley rats. The model was evaluated for up to 8 weeks with a transit-time flow meter, a laser Doppler perfusion imager, microspheres, arteriography and histology. It was found to be well tolerated with low mortality and perfusion in the foot skin was reduced up to 8 weeks, while collaterals were visible after 2 weeks. Histologic signs of ischemia were seen for up to 4 weeks. This rat model of severe limb ischemia at rest lasts up to 8 weeks and seems well suited for longitudinal studies of the pathophysiology of limb ischemia and healing mechanisms like angio- and arteriogenesis.

Angiography↗

PubMedDate not supplied

TP53 mutations do not correlate with locoregional recurrence in stage I tongue carcinomas.

BACKGROUND: The abrogation of the TP53 gene is considered to play a central role in the development of human cancers. Exons 5-8 harbor mutations most frequently, mainly of the missense type, resulting in accumulation of the p53 protein. The importance of these alterations as prognostic factors, are issues of controversy. MATERIAL AND METHODS: Thirty-four patients suffering from stage I tongue carcinoma had been treated with a local surgical excision of the tumor. Seventeen patients had developed a local recurrence in the tongue or cervical (regional) metastases while 17 patients, matched for age and gender to the former group, had no recurring disease within follow-up. Protein p53 was detected through immunohistochemical (IHC) analysis using antibody CM1. Exons 5-8 of the TP53 gene were amplified through the Polymerase Chain Reaction (PCR). The presence of mutations analyzed by CDGE (Constant Denaturant Gel Electrophoresis) and detected mutations were subjected to sequencing. RESULTS: 20 out of 34 tumors (59%) showed mutated TP53, 18 tumors were IHC p53 positive, but the correlation between CDGE and IHC was only 56%. Sequencing of the gene was possible in 8 cases. CONCLUSIONS: Neither the presence of mutations nor immunostaining had any impact on the risk of recurrence expressed as life-table analysis of time to recurrence.

Adult↗