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R F Kefford

Publications and source records attributed to R F Kefford.

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↗
PubMed2003

Adjuvant therapy of cutaneous melanoma: the interferon debate.

Despite the use of a variety of cytotoxic and immunotherapeutic agents in adjuvant trials in patients following resection of high-risk early cutaneous melanoma, only interferon-alpha2b (IFN-alpha) has shown reproducible efficacy. High-dose IFN-alpha (HDI) is superior to observation in prolonging relapse-free survival. There is still no formal proof of a statistically significant advantage of HDI in prolonging overall survival. For this reason the continued use of observation-only control arms is justified and desirable in adjuvant melanoma trials, and, wherever possible, patients with resected high-risk and intermediate-risk melanoma should be entered on these studies. The toxicity of HDI is high, but the majority of patients complete treatment with dose modification and nearly all toxicity is rapidly reversible. There is now a useful body of information on the supportive care of patients receiving HDI, and data on cost and quality-adjusted time without symptoms and toxicity (Q-TwiST) to support its use in certain high-risk patients. Interim results from a trial of intermediate-dose IFN-alpha are promising. These, and ongoing studies of pegylated IFN-alpha, and of shorter induction-only HDI promise refinements in treatment which may improve efficacy:toxicity ratios.

Antineoplastic Agents↗
PubMed2001

A melanoma-associated germline mutation in exon 1beta inactivates p14ARF.

The INK4a/ARF locus encodes the cyclin dependent kinase inhibitor, p16(INK4a) and the p53 activator, p14ARF. These two proteins have an independent first exon (exon 1alpha and exon 1beta, respectively) but share exons 2 and 3 and are translated in different reading frames. Germline mutations in this locus are associated with melanoma susceptibility in 20-40% of multiple case melanoma families. Although most of these mutations specifically inactivate p16(INK4a), more than 40% of the INK4a/ARF alterations located in exon 2, affect both p16(INK4a) and p14ARF. We now report a 16 base pair exon 1beta germline insertion specifically altering p14ARF, but not p16(INK4a), in an individual with multiple primary melanomas. This mutant p14ARF, 60ins16, was restricted to the cytoplasm, did not stabilize p53 and was unable to arrest the growth of a p53 expressing melanoma cell line. This is the first example of an exon 1beta mutation that inactivates p14ARF, and thus implicates a role for this tumour suppressor in melanoma predisposition.

Adult↗
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

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↗
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↗
PubMed1995

The spectrum of in vitro radiosensitivity in four human melanoma cell lines is not accounted for by differential induction or rejoining of DNA double strand breaks.

PURPOSE: Radioresistance is a significant clinical problem in advanced malignant melanoma and many melanoma cell lines show a radioresistant acute x-ray survival response in vitro. Given that the DNA double strand break is the lesion most closely correlated with x-ray induced cell lethality, differences in the induction and rejoining of these lesions may account for the radioresistance of some human melanoma cell lines. METHODS AND MATERIALS: The above hypothesis was tested using pulsed field gel electrophoresis to measure x-ray induced DNA double strand break induction and rejoining in four human melanoma cell lines: MM138, MM170, MM96-L and HT 144. RESULTS: The MM138, MM170 and MM96-L cell lines were characterized in vitro by low alpha/beta ratios and broad x-ray survival curve shoulders. MM138 and MM170 were the most radioresistant and MM96-L had intermediate sensitivity. In contrast, HT144 was markedly x-ray sensitive, despite retaining a shoulder and like the other lines, having a low alpha/beta ratio. There were no significant differences in DNA double strand break induction between the cell lines, and thus no correlation existed between DNA double strand break induction and radiosensitivity. Consistent with the shoulders on the x-ray survival curves, all four cell lines showed efficient DNA double strand break rejoining. Highly efficient DNA double strand break rejoining could account for the radioresistance of one of the melanoma lines (MM138). For example, MM138 had rejoined 50% of the induced DNA double strand breaks by 5.5 min compared to 13-17 min for the other three cell lines. The development of postirradiation apoptosis was effectively excluded as the cause of the marked radiosensitivity of the HT144 cell line. CONCLUSION: Other factors (such as lesion repair fidelity or differential lesion tolerance) underlie the differences in the intrinsic radiosensitivity between these melanoma cell lines.

Apoptosis↗
PubMed1994

Filgrastim in patients with chemotherapy-induced febrile neutropenia. A double-blind, placebo-controlled trial.

OBJECTIVE: To determine if filgrastim (recombinant human methionyl granulocyte colony-stimulating factor) used in addition to standard inpatient antibiotic therapy accelerated recovery from infection associated with chemotherapy-induced neutropenia. DESIGN: Randomized, double-blind, placebo-controlled trial. SETTING: Hematology and oncology wards of four teaching hospitals. PATIENTS: 218 patients with cancer who had fever (temperature > 38.2 degrees C) and neutropenia (neutrophil count < 1.0 x 10(9)/L) after chemotherapy. INTERVENTION: Patients were randomly assigned to receive filgrastim (12 micrograms/kg of body weight per day) (n = 109) or placebo (n = 107) beginning within 12 hours of empiric therapy with tobramycin and piperacillin. Patients received treatment and remained in the study until the neutrophil count was greater than 0.5 x 10(9)/L and until 4 days without fever (temperature < 37.5 degrees C) had elapsed. MEASUREMENTS: Days of neutropenia and fever and days in the study (hospitalization); time to resolution of fever and febrile neutropenia; and frequency of the use of alternative antibiotics. RESULTS: Compared with placebo, filgrastim reduced the median number of days of neutropenia (3.0 compared with 4.0 days of a neutrophil count of < 0.5 x 10(9)/L; P = 0.005) and the time to resolution of febrile neutropenia (5.0 compared with 6.0 days; P = 0.01) but not days of fever (3.0 days for both groups). The frequency of the use of alternative antibiotics was similar in the two groups (46% compared with 41%; P = 0.48). The median number of days patients were hospitalized while on study was the same (8.0 days; P = 0.09); however, filgrastim decreased the risk for prolonged hospitalization (> 11 days, 4th quartile) by half (relative risk, 2.1 [95% CI, 1.1 to 4.1]; P = 0.02). In exploratory subset analyses, filgrastim appeared to provide the greatest benefit in patients with documented infection and in patients presenting with neutrophil counts of less than 0.1 x 10(9)/L. CONCLUSIONS: Filgrastim treatment used with antibiotics at the onset of febrile neutropenia in patients with cancer who have received chemotherapy accelerated neutrophil recovery and shortened the duration of febrile neutropenia.

Adolescent↗