PubMed Health⌕ Search

Biomedical subjects

G J Mann

Publications and source records attributed to G J Mann.

At least 19 recordsLinked to original sources
PubMed2005

Impaired inhibition of NF-kappaB activity by melanoma-associated p16INK4a mutations.

The cyclin-dependent kinase inhibitor p16INK4a has been identified as tumor suppressor and melanoma predisposition gene. While its cell cycle inhibitory ability is important in protecting cells from uncontrolled growth and possible tumor formation, other functions of p16INK4a are likely to contribute to its nature as a tumor suppressor. p16INK4a binding and inhibition of the transcription factor NF-kappaB has been shown and is consistent with the reports of abnormally increased NF-kappaB activity in various cancers including melanoma. Here, we present evidence that wild type p16INK4a binds to the NF-kappaB subunit RelA more efficiently than melanoma-associated p16INK4a mutations. Furthermore, whereas wild type p16INK4a strongly inhibits NF-kappaB transcriptional activity, a subset of melanoma-associated p16INK4a mutants show reduced NF-kappaB inhibitory function. p16INK4a does not affect NF-kappaB nuclear translocation or DNA binding, suggesting a mechanism that reduces NF-kappaB transactivation activity.

Active Transport, Cell Nucleus↗
PubMed2001

Mutations in the INK4a/ARF melanoma susceptibility locus functionally impair p14ARF.

The INK4a/ARF locus encodes two cell cycle regulatory proteins, the cyclin-dependent kinase inhibitor, p16(INK4a), and the p53 activator, p14(ARF). Germline mutations in this locus are associated with melanoma susceptibility in 20-40% of multiple case melanoma families. Many of these mutations specifically impair p16(INK4a), whereas mutations uniquely targeting p14(ARF) are rare. Nevertheless, the importance of p14(ARF) has not been excluded because more than 40% of INK4a/ARF alterations affect p16(INK4a) and p14(ARF). We now report that p14(ARF) is functionally impaired in melanoma kindreds carrying INK4a/ARF mutations. Of the seven INK4a/ARF mutations tested, three altered the subcellular distribution of p14(ARF) and diminished the ability of p14(ARF) to activate the p53 pathway. This work establishes the importance of p14(ARF) in melanoma predisposition.

Amino Acid Sequence↗
PubMed2001

The intronic G13964C variant in p53 is not a high-risk mutation in familial breast cancer in Australia.

BACKGROUND: Mutations in BRCA1 and BRCA2 account for approximately 50% of breast cancer families with more than four affected cases, whereas exonic mutations in p53, PTEN, CHK2 and ATM may account for a very small proportion. It was recently reported that an intronic variant of p53--G13964C--occurred in three out of 42 (7.1%) 'hereditary' breast cancer patients, but not in any of 171 'sporadic' breast cancer control individuals (P = 0.0003). If this relatively frequent occurrence of G13964C in familial breast cancer and absence in control individuals were confirmed, then this would suggest that the G13964C variant plays a role in breast cancer susceptibility. METHOD: We genotyped 71 familial breast cancer patients and 143 control individuals for the G13964C variant using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis. RESULTS: Three (4.2%; 95% confidence interval [CI] 0-8.9%) G13964C heterozygotes were identified. The variant was also identified in 5 out of 143 (3.5%; 95% CI 0.6-6.4%) control individuals without breast cancer or a family history of breast cancer, however, which is no different to the proportion found in familial cases (P = 0.9). CONCLUSION: The present study would have had 80% power to detect an odds ratio of 4.4, and we therefore conclude that the G13946C polymorphism is not a 'high-risk' mutation for familial breast cancer.

Adult↗
PubMed2001

Responses to ultraviolet-B in cell lines from hereditary melanoma kindreds.

Ultraviolet-B (UV-B) triggers a cascade of events involving cell cycle control genes leading ultimately to DNA repair or apoptosis. The hypothesis examined here is that the genetic abnormality predisposing to melanoma affects the ability of the cell to respond appropriately to UV-B, so favouring mutagenesis. Epstein-Barr virus-transformed lymphoblastoid cell lines from hereditary melanoma kindreds were irradiated with UV-B, and changes in p53, p21 and Bcl-2 expression and cell cycle phase distribution were analysed. Twenty-two cell lines were tested: 12 carriers of melanoma susceptibility and 10 non-carriers (unaffected first degree relatives). At 24 h after irradiation with 50 J/m2, 15 of the 22 cell lines showed a rise in G2/M. After 400 J/m2, all the cell lines showed a reduction or loss of G2/M and 17 of the 22 showed an S phase delay. More carriers than noncarriers of melanoma susceptibility showed significant S phase delay after 50 J/m2 (seven out of 12 carriers versus two out of 10 non-carriers). Six of the 10 pairs (carrier versus non-carrier) tested showed discordant cell cycle responses; however the nature of the difference was not universal. Bcl-2 reduction was seen 4 h post-irradiation in all the carriers and non-carriers. The p53 and p21 responses, although showing some individual variations, were not related to carrier status. These results show individual variations in response to UV-B irradiation among cell lines from the members of hereditary melanoma kindreds, but no consistent differences between carriers and non-carriers of melanoma susceptibility.

Apoptosis↗
PubMed2001

Functional impairment of melanoma-associated p16(INK4a) mutants in melanoma cells despite retention of cyclin-dependent kinase 4 binding.

PURPOSE: Melanoma-associated germ-line mutations affecting the tumor suppressor and cyclin-dependent kinase (CDK) inhibitor, CDKN2A/p16(INK4a) have been identified in >100 melanoma-prone families. To predict the melanoma risk for carriers of specific mutations, it is useful to test the function of the mutant proteins in biochemical assays; however, it is unclear how well these results correlate with their cellular effects. We examined the relationship between loss of CDK binding by mutant proteins and various measures of cellular growth in melanoma cells. EXPERIMENTAL DESIGN: The cellular activities of four melanoma-associated p16(INK4a) mutations (Arg24Pro, Ala36Pro, Met53Ile, and Val126Asp) were compared by use of inducible expression in stably transfected melanoma cells, deficient in expression of the endogenous protein, and compared with their ability to bind CDK4. RESULTS: The cell cycle-inhibitory activity of all of the mutants was profoundly reduced, and partially retained capacity for CDK4 binding in functional assays did not correlate with significant preservation of cell cycle-regulatory function. CONCLUSION: Testing of p16(INK4a) interactions with CDKs in protein-binding assays is an unreliable predictor of mutant p16(INK4a) function in cells. In addition to exhibiting reduced stability, these mutant proteins may also be defective in interaction with cellular targets other than CDKs.

Cell Division↗
PubMed2001

X inactivation, DNA deletion, and microsatellite instability in common acquired melanocytic nevi.

We have investigated several molecular characteristics of common acquired melanocytic nevi to clarify their relationship to malignant melanoma, which is characterized by clonality and the progressive accumulation of DNA deletions. Twenty-four common acquired nevi were subjected to analysis for loss of heterozygosity at four loci on chromosome 9p and six loci on 10q that are commonly deleted in melanoma, but no deletions were seen. X inactivation analysis was performed in lesions from females, using the methylation-sensitive restriction HpaII site in the CAG microsatellite repeat (HUMARA) in exon 1 of the androgen receptor (AR) gene. In 14 melanomas, 11 (92%) were confirmed to have skewed X inactivation, consistent with monoclonality, as were 16 (80%) of 20 benign nevi. One nevus (5%) and 4 (33%) of 12 melanomas also showed loss of heterozygosity at HUMARA. One nevus showed an additional allele, consistent with low level microsatellite instability, at one of the 11 loci that were examined. Common melanocytic nevi, therefore, arise by apparently clonal proliferation, but they do not share chromosomal deletions that are characteristic of melanoma. However, skewed X inactivation patterns were seen in some samples of adjacent microdissected normal epidermis.

Chromosomes, Human, Pair 10↗
PubMed2000

Two arginine rich domains in the p14ARF tumour suppressor mediate nucleolar localization.

The INK4a/ARF locus encodes two distinct tumour suppressors, p16INK4a and p14ARF, that regulate cell cycle progression via the pRB and p53 pathways, respectively. The ARF protein inhibits hdm2 activity, leading to the stabilization of the p53 tumour suppressor and cell cycle inhibition. The amino-terminal domain of human p14ARF and of the mouse homologue, p19ARF, is sufficient for these effects. This domain is also sufficient for the nucleolar localization of the mouse ARF protein. In contrast, we show that the human ARF protein requires two arginine rich domains, one in the amino- and the other in the carboxy-terminus, for nucleolar targeting. The amino-terminal nucleolar-targeting domain of p14ARF is also important for ARF-hdm2 binding and cell cycle inhibition. The carboxy-terminal p14ARF nucleolar localization domain lies within the shared INK4a/ARF exon 2, and is mutated in a small number of melanoma-prone kindreds. The INK4a/ARF exon2-mutations could affect the function of both the p16INK4a and p14ARF tumour suppressors. Oncogene (2000).

Amino Acid Sequence↗
PubMed2000

Mutation screening of the CDKN2A promoter in melanoma families.

Germline mutations of CDKN2A, at 9p21, are responsible for predisposition to melanoma in some families. However, evidence of linkage to 9p21 has been demonstrated in a significant proportion of kindreds with no detectable mutations in CDKN2A. It is possible that mutations in noncoding regions may be responsible for predisposition to melanoma in these families. We have analyzed approximately 1 kb of the CDKN2A promoter upstream of the start codon in an attempt to identify causal mutations in 107 melanoma families. Four sequence variants were detected. Two of these (A-191G and A-493T) did not segregate with disease and were present in a control population at a comparable frequency, indicating that they are unlikely to predispose to melanoma. The A-493T variant appeared to be in linkage disequilibrium with the previously described CDKN2A polymorphism Ala148Thr. The variant G-735A was detected in the control population, but segregation of this variant with melanoma within families could not be discounted. The fourth variant (G-34T), located in the 5' UTR, creates an aberrant initiation codon. This variant appeared to segregate with melanoma and was not detected in a control population. G-34T has recently been identified in a subset of Canadian melanoma families and was concluded to be associated with predisposition to melanoma. The creation of an aberrant initiation site in the 5' UTR may have an important role in carcinogenesis in a small percentage of families; however, mutations in the CDKN2A promoter appear to have a limited role in predisposition to melanoma.

Adolescent↗
PubMed2000

CDKN2A mutation and deletion status in thin and thick primary melanoma.

Human melanoma cell lines and tumor tissue from familial and sporadic melanomas have frequent, nonrandom chromosomal breaks and deletions on chromosome 9p21, a region that includes the tumor suppressor gene CDKN2A/p16INK4A. Germ-line mutations within this gene have been observed in some familial melanoma kindreds, but somatic mutation in sporadic primary melanoma is infrequent. Thirty-nine archival, paraffin-embedded, sporadic, primary cutaneous malignant melanomas (20 >3-mm-thick and 19 <0.75-mm-thick cases) were examined for mutations of the CDKN2A gene using single-strand conformational polymorphism analysis and direct sequencing. No mutations were detected. Loss of heterozygosity for the 9p21 microsatellite marker D9S942 was detected in 6 of 17 informative thick lesions (35%) but 0 of 18 thin lesions (P = 0.006). These results support other studies indicating that intragenic mutation is an infrequent mechanism of CDKN2A inactivation in primary melanoma. The finding of loss of heterozygosity for the 9p21 microsatellite D9S942 in thick but not thin primary melanoma suggests that deletion or inactivation of CDKN2A or other tumor suppressor gene(s) at this locus is involved in the progression rather than initiation of sporadic malignant melanoma.

Chromosomes, Human, Pair 9↗
PubMed1999

Multiple abnormalities of the p16INK4a-pRb regulatory pathway in cultured melanoma cells.

The retinoblastoma protein (pRb) pathway is critical in regulating the G1 phase of the cell cycle and it is frequently disrupted in human cancers. Components of the pRb pathway which are often altered in tumour progression include the INK4 cyclin-dependent kinase (CDK) inhibitors p16INK4a/ CDKN2A and p15INK4b/CDKN2B, CDK4, D-type cyclins and pRb. Several of these components were studied in a series of cultured melanoma cell lines in order to determine the frequency and spectrum of genetic alterations and to define targets for potential gene transfer studies. Also studied were the p16INK4a alternate transcript (p14ARF) and the p21(waf1) CDK inhibitor. The majority of the melanoma cell lines tested (13 out of 17; 76%) carried mutated (two), deleted (nine) or silenced (two) p16(INK4a). CDK4 was mutated or overexpressed in two melanoma cell lines with homozygously deleted CDKN2A and CDKN2B genes. This suggests that the selective growth advantages afforded by CDKN2A inactivation and CDK4 insensitivity are distinct and may involve the mediation of other CDK inhibitors or CDKs.

Blotting, Northern↗
PubMed1999

Normal repair of ultraviolet radiation-induced DNA damage in familial melanoma without CDKN2A or CDK4 gene mutation.

Excessive sun exposure and family history are strong risk factors for the development of cutaneous melanoma. Inherited susceptibility to this type of skin cancer could therefore result from constitutively impaired capacity to repair ultraviolet (UV)-induced DNA lesions. While a proportion of familial melanoma kindreds exhibit germline mutations in the cell cycle regulatory gene CDKN2A (p16INK4a) or its protein target, cyclin-dependent kinase 4 (CDK4), the biochemical basis of most familial melanoma is unknown. We have examined lymphoblastoid cell lines from melanoma-affected and unaffected individuals from large hereditary melanoma kindreds which are not attributable to CDKN2A or CDK4 gene mutation. These lines were tested for sensitivity of clonogenic growth to UV radiation and for their ability to repair transfected UV-damaged plasmid templates (host cell reactivation). Two of seven affected-unaffected pairs differed in colony survival after exposure to UVB radiation; however, no significant differences were observed in the host-cell reactivation assays. These results indicate that melanoma susceptibility genes other than CDKN2A and CDK4 do not impair net capacity to repair UV-induced DNA damage.

Chloramphenicol O-Acetyltransferase↗
PubMed1999

CDKN2A (P16(INK4a)) and CDK4 mutation analysis in 131 Australian melanoma probands: effect of family history and multiple primary melanomas.

Mutation analysis of two genes involved in melanoma susceptibility (CDKN2A/p16(INK4a) and CDK4) was undertaken in 131 probands with a family history of melanoma. Screening of all three exons of CDKN2A and exon 2 of CDK4 by single-strand conformation polymorphism (SSCP) analysis and/or direct sequencing identified a total of 10 different CDKN2A germline mutations, including 6 not previously described in the germline. All but one has been previously proven to, or is likely to, affect the structure and function of p16(INK4a). The incidence of CDKN2A mutation was 8.4% (11/131), but was significantly higher in families with three or more cases of melanoma (10/66, 15.1%) than in those in which only two relatives were affected (1/65, 1.5%). The incidence of CDKN2A mutation was also higher in families with three or more cases of melanoma and at least one member with multiple primary melanomas (6/19, 31.6%) than in similar families without multiple primary melanomas (4/47, 8.5%). One novel CDK4 variant of uncertain significance was found in a kindred that also carries a CDKN2A mutation. Genes Chromosomes Cancer 25:339-348, 1999.

Adolescent↗
PubMed1998

10q deletions in metastatic cutaneous melanoma.

Cytogenetic and allelic deletion studies have indicated that the loss of distal chromosome 10q may be a frequent and early event in melanoma tumorigenesis. We have studied nine polymorphic markers spanning 56 cM of this region in 27 advanced melanomas and find that half exhibited loss of the entire region, but none had more limited deletions. Because all these tumors had a codeletion of 9p, the 10q deletion event is likely to impair a pathway other than the cyclin-dependent kinase-mediated phosphorylation of the retinoblastoma protein.

Chromosome Deletion↗
PubMed1997

Differential expression of p16INK4a and p16beta transcripts in B-lymphoblastoid cells from members of hereditary melanoma families without CDKN2A exon mutations.

Mutations in the CDKN2A (p16INK4a) tumour suppressor gene on chromosome 9p21 are associated with inherited predisposition to melanoma, yet some 9p-linking hereditary melanoma families show no mutations in this gene. Splicing of CDKN2A exons 2 and 3 to an alternative first exon produces a transcript (p16beta) encoding a protein with cell cycle regulatory properties. We have analysed allele-specific expression levels of both the p16INK4a and p16beta transcripts in B-lymphoblastoid cells from 18 members of hereditary melanoma kindreds including four unrelated control individuals. In 15 of the 18 individuals examined, steady-state levels of each transcript either originated equally from each parental chromosome, or one parental chromosome was dominant for both transcripts. However, in three affected members of two 9p-linking hereditary melanoma kindreds, without exonic CDKN2A mutations, this pattern of coordinate expression was disrupted. In these individuals there was underexpression of the p16beta transcript, relative to the p16INK4a transcript, from the chromosome segregating with disease susceptibility. Loss of coordinate expression of the p16INK4a and p16beta transcripts may be an alternative genetic basis for melanoma susceptibility in certain 9p-linking kindreds.

Alternative Splicing↗
PubMed1997

Linkage analysis of familial melanoma and chromosome 6 in 14 Australian kindreds.

CDKN2A (9p21) and CDK4 (12q13) have been identified as melanoma susceptibility genes in certain familial melanoma (FM) kindreds. There remain other FM families, however, for which there is little or no evidence for linkage of melanoma to these loci. Other loci may be involved in susceptibility to this malignancy. Chromosome 6 is deleted or rearranged in 66% of melanomas and has been targeted by several studies in an attempt to identify chromosomal regions associated with initiation or progression of melanoma. Previous studies of familial melanoma and chromosome arm 6p reported evidence suggestive of linkage for markers flanking the HLA complex. We have carried out genetic linkage analysis in 14 Australian familial melanoma kindreds using 16 short tandem repeat polymorphism (STRP) markers spanning 6p23-6q27. Analysis by maximum likelihood and non-parametric (affected pedigree member) techniques showed no evidence of linkage of melanoma in this family set to chromosome 6 (two-point Zmax = 0.5 at theta = 0.2 for D6S285). Lod scores > 1.0 were obtained for the loci D6S285, D6S105, D6S265, D6S292, and D6S311 in three individual kindreds but these were insufficiently strong for formal heterogeneity testing to confirm that a chromosome 6-linked subset of families exists. These data imply little or no role for a major chromosome 6 melanoma susceptibility locus; however the possibility of such a locus remains open and warrants further investigation.

Alleles↗
PubMed1995

Analysis of the p16 gene, CDKN2, in 17 Australian melanoma kindreds.

CDKN2 has been implicated as a melanoma susceptibility gene in some kindreds with a family history of this disease. Mutation analysis of CDKN2 in 17 familial melanoma Australian kindreds revealed a paucity of exon mutations and none of the previously described disease-related mutations. One novel germline mutation was found in exon one, Arg24Pro, which segregates with melanoma in 1/17 kindreds. Two previously described polymorphisms, Ala148Thr and a base change at nucleotide 540 were detected and one novel polymorphism in the untranslated region of exon 3 (nucleotide 580) was also found. Together with other recent reports, these findings provide support for CDKN2 as a susceptibility locus for familial melanoma but suggest that other loci are involved in some hereditary melanoma kindreds.

Australia↗
PubMed1994

Purification, characterization, and localization of subunit interaction area of recombinant mouse ribonucleotide reductase R1 subunit.

Mammalian ribonucleotide reductase is a heterotetramer formed by the two non-identical homodimers proteins R1 and R2. We have succeeded in expressing the 90-kDa mouse R1 protein in Escherichia coli in an active, soluble form using the T7 RNA polymerase pET vector system. To avoid inclusion bodies, the bacteria were grown at 15 degrees C with minimal concentration of the inducer isopropyl-1-thio-beta-D-galactopyranoside. After a rapid purification procedure, approximately 20 mg of pure R1 protein were obtained per liter of bacterial culture. The concentrated R1 protein solution had a pinkish red color. Spectroscopy in combination with iron and labile sulfur analyses demonstrated that the color originated from an iron-sulfur complex. However, all attempts to demonstrate a function of this complex have been inconclusive. A comparison of the recombinant R1 protein with the corresponding protein purified from calf thymus showed no evidence for glycosylation. Circular dichroism spectroscopy indicated an alpha-helical content of 50%. A flexible COOH-terminal tail of 7 residues in the R2 protein was earlier shown to be essential for binding to the R1 protein. Using a peptide protection assay and photoaffinity labeling, we now show that the R2 protein tail interacts with a region close to the carboxyl terminus of the R1 protein.

Affinity Labels↗