Heritability of DNA-damage-induced apoptosis and its relationship with age in lymphocytes from female twins.

Apoptosis is a physiological form of cell death important in normal processes such as morphogenesis and the functioning of the immune system. In addition, defects in the apoptotic process play a major role in a number of important areas of disease, such as autoimmune diseases and cancer. DNA-damage-induced apoptosis plays a vital role in the maintenance of genomic stability by the removal of damaged cells. Previous studies of the apoptotic response (AR) to radiation-induced DNA damage of lymphoid cells from individuals carrying germline TP53 mutations have demonstrated a defective AR compared with normal controls. We have also previously demonstrated that AR is reduced as individuals age. Results from the current study on 108 twins aged 18-80 years confirm these earlier findings that the AR of lymphoid cells to DNA damage is significantly reduced with increasing age. In addition this twin study shows, for the first time, that DNA-damage-induced AR has a strong degree of heritability of 81% (95% confidence interval 67-89%). The vital role of DNA-damage-induced apoptosis in maintaining genetic stability, its relationship with age and its strong heritability underline the importance of this area of biology and suggest areas for further study.

Apoptosis, ageing and cancer susceptibility.

We have previously shown that peripheral blood lymphocytes (PBL) from individuals carrying a germline TP53 mutation show a dramatically reduced apoptotic response to radiation. As part of a study of this phenomenon, we also investigated apoptotic response in a series of breast cancer patients lacking TP53 mutations and in a control group of individuals without cancer. There was a significant reduction in mean apoptotic response with increasing age in all groups. These findings are consistent with a number of studies in rodents, which have demonstrated a reduction in DNA damage-induced apoptosis with increasing age. In addition, after adjusting for age, breast cancer patients showed significantly reduced apoptotic responses compared with normal controls (P=0.002). The odds ratio for breast cancer in women with an apoptotic response of <35%, compared with women with a response of >49%, was 6.42 (95% CI 1.68-24.6). The data further support the hypothesis that a reduction in apoptotic response to DNA damage with increasing age may play a significant role in the age-related increase in cancer.

Investigations on a clinically and functionally unusual and novel germline p53 mutation.

This report describes an individual with a rare choroid plexus papilloma in adulthood (age 29) after earlier having an osteosarcoma (age 22). The results from this study, and others, suggest that it may be advisable to consider the possibility of a germline p53 mutation in adults presenting with choroid plexus tumours. In the current study automated DNA sequencing of genomic DNA detected a novel germline 7 base pair insertion in exon 5 of the p53 gene in this patient. The alteration in frame would produce amino acid substitutions beginning with alanine to glycine at position 161 and a stop codon at position 182 in the mutated protein. Surprisingly two assays of p53 function gave apparently wild-type results on peripheral blood lymphocytes from this individual. These results led us to carry out more detailed functional tests on the mutant protein. The mutant allele was expressed either at very low levels or not at all in phytohaemagglutinin stimulated lymphocytes. Further, the mutant protein was completely non-functional in terms of its ability to transactivate a series of p53-responsive genes (p21(WAF1), bax, PIG3), to transrepress a target gene and to inhibit colony growth in transfected Saos-2 cells. However, surprisingly, data from irradiated peripheral blood lymphocytes and transfected Saos-2 cells, suggested that this truncated, mutant protein retains significant ability to induce apoptosis.

Mitotic counts provide additional prognostic information in grade II mammary carcinoma.

The ability to predict how long a patient diagnosed with breast cancer is likely to survive is still imprecise, despite numerous studies which have identified potential prognostic markers. The "established" markers such as nodal status, tumour size, and histological grade have been used for many years and certainly provide some degree of accuracy upon which treatment can be based. However, women with similar prognostic features can vary significantly in their outcome and very few of the newly identified markers provide information that is sufficiently useful to warrant the time and expense spent on their evaluation. In a cohort of 145 women, an assessment has been made of whether knowledge of the proliferative activity of grade II infiltrating ductal breast carcinomas can improve the accuracy of predicting clinical outcome for individual patients. Use of the mitotic count (MC), which was assessed as part of the grading system, enabled patients to be stratified into "good" and "bad" prognostic groups. The measurement of S-phase fraction using flow cytometry gave a similar result, but has the disadvantage that the technique requires specialized equipment. The evaluation of Ki-67 expression using immunohistochemistry was of no additional prognostic value in this defined group. It is proposed that MC, used once to establish grade, could be used again amongst the grade II tumours to improve the accuracy of prognosis and thus influence treatment strategies with minimal additional effort or expense.

p53 functional assays: detecting p53 mutations in both the germline and in sporadic tumours.

The tumour suppressor gene p53 is the gene most often reported to be mutated in clinical cancers with something like half of all tumours harbouring mutations. Further, many studies have suggested that p53 mutations have prognostic importance and sometimes are a significant factor in determining the response of tumours to therapy. The value of knowing the p53 status of individual tumours will increase if currently researched strategies aimed at developing p53-based treatment protocols come to fruition. There are quite a number of techniques used to detect p53 defects in both tumours and in the germline of cancer-prone families, although some of these methods are indirect and each has certain drawbacks. In this brief review we will discuss the value of two assays of p53 function as a means of detecting and partly characterizing p53 mutations. The two assays are the apoptotic assay, which measures the response of peripheral blood lymphocytes to radiation-induced DNA damage and the FASAY, a yeast based assay which assesses the ability of a given p53 protein to transactivate p53 target genes. Both of these assays are rapid, yielding results within 5 days. Further, they not only offer the possibility of detecting p53 mutations but also of characterizing a given mutation in terms of two of p53's most important functions, namely the induction of apoptosis and the transactivation of target genes.

Flow cytometric measurement of cell proliferation.

The simplest guide to cell proliferation that can be obtained by the use of flow cytometry is the S-phase fraction (SPF) calculated from DNA histograms. Measurement of such histograms was one of the earliest applications of flow cytometry, being first reported in the late 1960s. SPF is the fraction of cells in the S phase of the cell cycle and is broadly equivalent to a tritiated thymidine labeling index ([(3)H]TdR LI). The advantages of SPF as a proliferative index include that it can be obtained rapidly from fresh, frozen, or paraffin wax-embedded tissue without the need for radioactive chemicals. The disadvantages include the need to disaggregate solid tissues, thus losing tissue morphology, and the fact that, like mitotic index and [(3)H]TdR LI, SPF is only a crude proliferative index, which gives no details of the rate of cell proliferation. Flow cytometry offers a wide range of more sophisticated methods that allow more detailed analysis of the cell cycle and the rate of cell proliferation, and one such method involving bromodeoxyuridine (BrdUrd) labeling is described in this chapter. In the section on further reading, reference is made to sources that describe the combined measurement of DNA content and cell cycle related proteins (1,2). Also beyond the scope of this chapter are applications of flow cytometry that allow the measurement of cell death and differentiation, but references to these areas are also included (3).The literature on DNA flow cytometric studies is enormous, and a considerable number of such studies have looked at various aspects of the relationship between proliferation and metastasis.

The value of rapid functional assays of germline p53 status in LFS and LFL families.

We have tested two rapid assays of p53 function, namely the apoptotic assay and the FASAY as means of detecting germline p53 mutations in members of Li-Fraumeni and Li-Fraumeni-like families. Results of the functional assays have been compared with direct sequencing of all 11 exons of the p53 gene. The results show good agreement between the two functional assays and between them and sequencing. No false-positives or negatives were seen with either functional assay although the apoptotic assay gave one borderline result for an individual without a mutation. As an initial screen the apoptotic assay is not only rapid but inexpensive and very simple to perform. It would be expected to detect any germline defect that leads to loss of p53 function. The apoptotic assay could be ideal as a means of prescreening large numbers of samples and identifying those that require further investigation. The FASAY detects mutations in exons 4-10, is rapid and distinguishes between functionally important and silent mutations.

Improving the detection of p53 mutations in breast cancer by use of the FASAY, a functional assay.

The aim of this investigation was to examine the ability of the yeast-based functional assay, the functional analysis for the separation of alleles in yeast (FASAY), to detect p53 mutations in breast cancers when compared with immunohistochemistry and automated sequencing of the whole p53 gene (exons 1-11). To achieve this, all three methods were carried out on a cohort of aggressive breast tumors. In those tumors, in which the FASAY analysis indicated the presence of a mutation, cDNA was extracted from red yeast colonies and was sequenced to identify the base change in the p53 gene. The FASAY detected all 24 mutations found in the series of 48 tumors, whereas initial automated sequencing of genomic DNA detected 18/24 mutations. A second round of automated sequencing carried out using an independent source of genomic DNA detected mutations in 3 of the 6 tumors that originally appeared to lack a mutation in genomic DNA. All but 1 of the mutations originally missed by sequencing of genomic DNA were point mutations. Five mutations in this series (21%) were outside the commonly investigated exons 5-8, reinforcing the need to extend sequencing beyond this region. Of 24 tumors, 14 had strong immunohistochemical staining, and all 14 had p53 mutations; the majority of mutations missed by immunohistochemistry produced a truncated protein. Strong staining was not seen in tumors lacking a p53 mutation. The FASAY proved to be a rapid, reliable, and effective method for identifying those breast tumors harboring p53 mutations.

A patient with 17 primary tumours and a germ line mutation in TP53: tumour induction by adjuvant therapy?

We report the case history of a woman with a germ line mutation in the TP53 gene who developed 17 separate primary tumours. The incidence of new tumours rose steeply after adjuvant tamoxifen treatment for breast cancer and adjuvant vaginal vault radiotherapy for endometrial cancer. This increase could be due to cumulative genetic damage from environmental agents and the fact that the patient lived to the relatively late age of 60 years, or to a high inherent deleterious somatic mutation rate, which could represent the inability of cells from patients with TP53 mutations to repair therapy-induced genetic damage.

Prognostic significance of DNA ploidy, S-phase fraction, and P-glycoprotein expression in colorectal cancer.

BACKGROUND AND OBJECTIVES: Parameters that allow prediction of the disease course in colorectal cancer would aid the development of improved treatment strategies. For this reason, we evaluated the prognostic value of flow cytometric DNA ploidy and S-phase fraction (SPF) and P-glycoprotein (Pgp) expression in this type of tumor. METHODS: The prognostic significance of DNA ploidy, SPF, and Pgp expression on paraffin-embedded sections from 107 patients with colorectal carcinoma was determined. The mean follow-up was 36.6 months (range = 3-72 months). DNA ploidy and SPF were evaluated by flow cytometry and Pgp by immunohistochemistry using monoclonal antibody C219. The Cox regression model was used to adjust for several clinical and pathologic covariates. RESULTS: Of the 107 carcinomas examined, 44 (41.1%) were classified as DNA diploid and 63 (58.9%) as DNA aneuploid. DNA ploidy pattern was significantly related to tumor site (P = 0.010), tumor stage (P = 0.016), and vascular invasion (P = 0.015) but not to other clinicopathologic variables. Patients with DNA diploid tumors showed a better survival rate than did those with aneuploid tumors. After stage IV disease was excluded, patients with diploid tumors also presented a better disease-free and overall survival than did patients with aneuploid tumors. Mean SPF of the whole series was 13.5% (median = 11.3%, range = 1.4%-29.9%). Aneuploid tumors had a higher median SPF than did diploid tumors (17 vs. 6.2; P = 0.0001). SPF was only related significantly with tumor location (P = 0.026). In the multivariate analysis, SPF was a significant independent prognostic factor for overall survival (P = 0.01). When stage IV was excluded, SPF was also an independent prognostic variable for both disease-free (P = 0. 02) and overall (P = 0.01) survival. Of 107 tumors, 61 (57%) were positive for Pgp expression, but no relation was found between this and other clinicopathologic parameters. Pgp expression had no influence on survival. CONCLUSIONS: Our results suggest that flow cytometric DNA ploidy and SPF are significant and independent prognostic factors in patients with colorectal carcinoma, whereas Pgp expression is not.

The need for dynamic methods for measuring cell cycle perturbations: a study in radiation-treated lymphoblastoid cell lines of varying p53 status.

Reports on the p53-related cell cycle and apoptotic responses of EBV-transformed lymphoblastoid cell lines to DNA damage have led to some confusion. This may be due to differences in the nature of the specific p53 mutations under examination, but it can also be partly attributed to methodological and analytical problems (e.g. the inappropriate use of static DNA histograms for cell cycle analysis). Taking seven lymphoblastoid cell lines derived from both normal individuals and Li-Fraumeni Syndrome/Li-Fraumeni-Like (LFS/LFL) patients of differing p53 status, we completed a detailed study of radiation-induced cell cycle perturbations. Using BrdUrd pulse labelling and flow cytometry it was found that, regardless of p53 status, the cells did not arrest in G1 despite all of the lines showing p53 upregulation 3 hours postirradiation. The irradiated cells did, however, show a general slowing both in S-phase entry from G1 and in movement through S-phase. These facts would not have been apparent from the analysis of static DNA histograms. The problems with the use of static methods to assess changes in the dynamics of cell cycle progression apply not only to studies involving EBV-transformed cell lines, but also to a wide range of investigations into the molecular control of cell proliferation.

Radiation-induced G1 arrest is not defective in fibroblasts from Li-Fraumeni families without TP53 mutations.

Radiation-induced G1 arrest was studied in four classes of early passage skin fibroblasts comprising 12 normals, 12 heterozygous (mut/wt) TP53 mutation-carriers, two homozygous (mut/-) TP53 mutation-carriers and 16 strains from nine Li-Fraumeni syndrome or Li-Fraumeni-like families in which no TP53 mutation has been found, despite sequencing of all exons, exon-intron boundaries, 3' and 5' untranslated regions and promoter regions. In an assay of p53 allelic expression in yeast, cDNAs from these non-mutation strains behaved as wild-type p53. Using two different assays, we found G1 arrest was reduced in heterozygous strains with mis-sense mutations and one truncation mutation, when compared to the range established for the normal cells. Heterozygous strains with mutations at splice sites behaved like normal cells, whilst homozygous (mut/-) strains showed either extremely reduced, or no, arrest. Strains from all nine non-mutation families gave responses within the normal range. Exceptions to the previously reported inverse correlation between G1 arrest and clonogenic radiation resistance were observed, indicating that these phenotypes are not strictly interdependent.

Characterization of p53 oligomerization domain mutations isolated from Li-Fraumeni and Li-Fraumeni like family members.

p53 is a tumour suppressor gene which functions as a transcription factor to upregulate genes for growth arrest and apoptosis following DNA damage. p53 mutations are associated with Li-Fraumeni and Li-Fraumeni like syndromes. Recently mutations of the oligomerization domain have been isolated from an LFS and an LFL family affecting respectively codon 344 (Leu to Pro) and 337 (Arg to Cys). The present study was designed to determine the affect of these mutations on the function of p53 protein. p53 344 Leu to Pro existed only in a monomeric form and could not bind to DNA. It was inactive at inducing apoptosis, transactivating luciferase from a bax promoter and inhibiting cell growth. In contrast, p53 337 Arg to Cys could form tetramers and could bind to DNA. However, p53 337 Arg to Cys was not fully active and could only induce apoptosis, transactivate luciferase from a bax promoter and inhibit cell growth with approximately 60% of the ability of wild-type p53. Both mutant proteins had reduced ability to bind to MDM2, p53 337 Arg to Cys being more reduced than p53 344 Leu to Pro. These results indicate that point mutations in the oligomerization domain can disrupt p53 function. In addition, the value of LFS and LFL families for the further understanding of the biological and biochemical properties of p53 is demonstrated.

Genetic and functional studies of a germline TP53 splicing mutation in a Li-Fraumeni-like family.

We report an extensive Li-Fraumeni-like family in which there is an unusual spectrum of tumours at relatively late onset. A germline TP53 splice donor mutation in exon 4 is present in all affected family members available for testing. The mutation abolishes correct splicing of intron 4 and techniques of RT-PCR have identified three different aberrant transcripts from the mutant TP53 allele. Using the yeast functional assay to analyse transcripts in cells from a number of family members with the mutant allele, TP53 appears wild-type. Functional studies have been carried out on cells from patients with and without cancer who carry the germline mutation, and on cells from unaffected individuals from the same family who do not carry the mutation. Using a number of functional endpoints known to distinguish between cells carrying mutant or wild-type TP53 alleles, we were unable to discriminate normal (wt/wt) from heterozygous (wt/mut) cells by lymphocyte apoptosis and fibroblast survival following low dose rate ionising radiation exposure. However germline mutation carriers show increased sensitivity to radiation-induced chromosome damage in the G2 phase of the cell cycle, and decreased transient and permanent G1 arrest. These studies demonstrate the importance of fully characterising the effects of TP53 germline mutations, and may explain some of the phenotypic features of this family.

Two functional assays employed to detect an unusual mutation in the oligomerisation domain of p53 in a Li-Fraumeni like family.

Previous investigations of a Li - Fraumeni like family (Barnes et al., 1992) demonstrated that both the proband and her mother had elevated p53 protein levels in both tumour tissue and normal tissue at sites distant from the tumour, although no mutation was found in the p53 gene. In the present study two recently described functional assays for p53, an apoptotic assay and the functional assay for the separation of alleles in yeast (FASAY), have been employed to study the functional activity of p53 from this patient. The results of the apoptotic assay demonstrated that this patient had a p53 functional defect and the FASAY result suggested that this defect was in fact a germline mutation of the p53 gene. A point mutation of codon 337, which results in an amino acid substitution of a cysteine for an arginine, was demonstrated initially in cDNA and was confirmed by sequencing of genomic DNA. This is an unusual mutation as it is in the oligomerisation domain of p53, in contrast to the majority of p53 mutations which are in the core DNA binding domain. This mutation results in a protein which still retains partial transactivational activity in the FASAY. The mutation of codon 337 is only the second reported case of a germline missense mutation occurring in the oligomerisation domain of p53.

P53, apoptosis, and breast cancer.

Wild-type p53 is a tumor suppressor gene that plays a central role in maintaining the genetic integrity of the cell by preventing cells with damaged DNA from further proliferation. Mutation and deletion of p53 are the most common genetic defects seen in clinical cancer. About 40% of breast carcinomas show high levels of stabilized, often mutant, p53 protein in their cells as detected by immunohistochemistry. p53-related defects in tumor cells correlate with a poor prognosis and may also indicate a poor response to chemotherapy. In experimental systems, the p53 status of cells is important in determining their sensitivity to radiation and chemotherapeutic drugs. Cells with functional p53 die by apoptosis, whilst similar cells lacking p53 function continue to proliferate, perpetuating potentially oncogenic mutations. Not only may p53 status be a marker of the biological aggressiveness of individual tumors and of their likely response to therapy, but restoration of normal p53 function is itself already a goal of cancer therapy.

Myxofibrosarcoma. Clinicopathologic analysis of 75 cases with emphasis on the low-grade variant.

Myxofibrosarcoma is one of the most common sarcomas in the extremities of elderly patients. We analysed the clinicopathologic features in a series of 75 patients. All patients were adults (range, 22-91 years; median, 66 years) with an approximately equal incidence in men and women. Thirty-five tumors arose in the lower and 25 in the upper extremities, nine on the trunk, two each in the retroperitoneum and the head and neck region, and one each in the pelvis and penis. Forty-eight cases (69.5%) were located in dermal or subcutaneous tissues. Distinctive histologic features included the following: a commonly nodular growth pattern; a myxoid matrix containing elongated, curvilinear capillaries; and fusiform, round or stellate tumor cells with indistinct cell margins, slightly eosinophilic cytoplasm, and hyperchromatic atypical nuclei. These lesions varied from a hypocellular, mainly myxoid, and purely spindle-cell appearance (low-grade neoplasms) to high-grade, pleomorphic (malignant fibrous histiocytoma-like) lesions with multinucleated giant cells, high mitotic activity, and areas of necrosis. Immunohistochemistry in 44 cases revealed only vimentin and occasional actin positivity. Ultrastructurally, tumor cells had a fibroblastic phenotype. DNA flow cytometry and proliferation analysis showed an association between aneuploidy and histologic grade. An average follow-up of 45 months (range, 5-300 months) in 60 cases has revealed local recurrence in 33 cases (54%). Thirteen patients developed metastases, and 13 tumor-related deaths occurred. A short interval to first local recurrence was associated with poor clinical outcome. The rate of local recurrence was independent of histologic grade, but only intermediate and high-grade neoplasms metastasized. The depth of the primary lesion did not influence the incidence of local recurrence. However, in deep-seated neoplasms, the incidence of metastases was higher and the percentage of tumor-related deaths was twice as high as in superficially located lesions, reflecting the fact that deep-seated lesions tended to be higher-grade, larger tumors. Myxofibrosarcoma tends to become progressively higher grade in recurrences, as demonstrated in five cases in our series. The poorly recognized low-grade myxofibrosarcoma is emphasized, as proper diagnosis and treatment and scrupulous follow-up are mandatory to avoid local recurrence and gradual tumor progression to a higher-grade neoplasm that may then metastasize.

An inbred colony of oncogene transgenic mice: diversity of tumours and potential as a therapeutic model.

Transgenic mice carrying the activated rat c-neu oncogene under transcriptional control of the MMTV promoter were backcrossed to BALB/c mice, with the aim of developing a model for cancer therapy. A total of 86 of 268 transgene-positive mice in the first five generations developed 93 histologically diverse tumours (median age of onset 18 months). The cumulative incidence of breast tumours at 24 months was 18%, and overall tumour incidence 31%. As well as expected c-neu expressing breast cancers, lymphomas and Harderian gland carcinomas developed. Virgin mice had fewer mammary tumours than those with two litters. Breast carcinomas metastasised to the lungs, and lymphomas were widely disseminated. The tumours showed a range of architectural patterns, which resembled human breast cancers or lymphomas. This diversity was reflected in S-phase fraction and aneuploidy. Breast tumours transplanted to nude mice showed variable responses to interferon (IFN)-alpha and gamma. A tumour transplanted to BALB/c mice responded to interleukin (IL)-12. There was significant decline in transgene positivity with successive generations. The diversity, histological and biological resemblance to human cancer suggests that the model has potential for evaluating novel therapies. However, further genetic and environmental manipulations are required to increase tumour incidence and decrease age of onset.

A possible screening test for inherited p53-related defects based on the apoptotic response of peripheral blood lymphocytes to DNA damage.

The cellular response, in terms of cell cycle arrest(s) and apoptosis, to radiation-induced DNA damage was studied. Experiments were performed on both mitogen-stimulated and resting peripheral blood lymphocytes (PBLs) from normal and cancer-prone (C-P) individuals. The C-P individuals comprised three patients carrying germline p53 mutations and three members of two families apparently without such mutations, but with an inherited defect which results in p53 deregulation as shown by high levels of stabilised p53 protein in normal tissues. Interestingly, mitogen-stimulated PBL, from both normal and C-P individuals failed to demonstrate a G1 arrest after gamma radiation. However, a clear difference was seen in the apoptotic response to DNA damage, of PBL from normal and C-P individuals; PBLs from C-P individuals with inherited p53-related defects had a reduced apoptotic response (P = 0.0003). There was a wide margin of separation, with no overlap between the two groups, supporting the possibility of using this altered apoptotic response as a screening test. This simple and rapid procedure could be used to identify those individuals in a C-P family who carry germline p53-related defects. The method appears to detect both individuals with p53 mutations and those apparently without mutations but with other p53-related defects.