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.

Heterogeneity in Li-Fraumeni families: p53 mutation analysis and immunohistochemical staining.

We have screened two families for constitutional TP53 mutations, one family with Li-Fraumeni syndrome and the other with features of this syndrome. We report a germline mutation in exon 7 of the TP53 gene in the family with "Li-Fraumeni-like" syndrome. The mutation occurred at codon 245 and causes a Gly-Ser amino acid change. It was inherited by both affected and unaffected subjects. Malignant tumours from all members of this family showed strong positive nuclear immunohistochemical staining with antibodies CM-1 and DO1, directed against TP53. In contrast, no constitutional TP53 mutations were found in a "classic" Li-Fraumeni family. In this family positive staining was seen in both malignant and normal tissues. These results support previous findings that variants of the Li-Fraumeni syndrome exist since not all LFS families carry TP53 germline mutations. Secondly, immunohistochemical positivity is not synonymous with an underlying mutation and is therefore inadequate as an exclusive diagnostic marker.

Genetic heterogeneity in familial malignant melanoma.

Following reports of linkage to chromosome 9p in families with malignant melanoma, we have been studying a series of UK families. Six families were selected with three or more cases of malignant melanoma. We have used a total of twelve markers mapping in the interval 9p13-p23 and constructed a set of haplotypes to study the inheritance of the disease chromosome. Of the six families, three were consistent with linkage to the short arm of 9, although their limited size precluded confirmation of linkage. One family was clearly unlinked, one family was either unlinked, or contains a sporadic case, or delimits the location of the melanoma gene, and one family was essentially uninformative. This is strong evidence for genetic heterogeneity in families with the malignant melanoma phenotype. We have also sequenced exon 2 of the recently identified candidate tumour suppressor gene, p16, in six individuals and found no evidence for germline mutations in this region of the p16 gene in our families with inherited malignant melanoma.

A mismatch recognition defect in colon carcinoma confers DNA microsatellite instability and a mutator phenotype.

We have analyzed spontaneous mutations in the adenine phosphoribosyltransferase gene of Chinese hamster clone B cells that exhibit a mutator phenotype because of defective mismatch binding. The mutator phenotype conferred increases in a limited number of mutational classes. The rates of transitions and most transversions were not significantly increased. The rates of A to T transversions and -2 frameshifts were strikingly elevated. These mutations were in repeated elements and 5 of 9 of the frameshifts were dinucleotide deletions in DNA sequences resembling microsatellites. The mismatch binding protein that is defective in the mutator line is a G-T mismatch recognition factor. Band-shift analysis indicated that the preferred substrate for the mismatch recognition protein is duplex DNA containing an extrahelical mono- or dinucleotide within repeated sequences. In agreement with a role in preventing minus frameshifts, a defective binding protein conferred an instability in clone B microsatellite DNA. A mismatch binding defect was also detected in Lo Vo, a human colorectal carcinoma cell line. Extracts of clone B or a second mismatch binding-deficient line, Raji-F12, did not complement Lo Vo extracts, indicating that these lines share a common defect. Our data provide a mechanistic explanation for the relation between defective mismatch recognition and the microsatellite instability of human colon cancer.

Human cardiac troponin T: identification of fetal isoforms and assignment of the TNNT2 locus to chromosome 1q.

The troponin complex is located on the thin filament of striated muscle and is composed of three component polypeptides: troponin T, troponin I, and troponin C. Three troponin T genes have been described on the basis of molecular cloning in humans and other vertebrates. These are expressed in a tissue-specific manner and encode the troponin T isoforms expressed in cardiac muscle, slow skeletal muscle, and fast skeletal muscle, respectively. Each of these genes is subject to alternative splicing, resulting in the production of multiple tissue-specific isoforms. We have cloned cDNAs encoding human cardiac troponin T from adult heart and have used these to demonstrate that multiple cardiac troponin T mRNAs are present in the human fetal heart, resulting from alternative splicing in the 5' coding region of the gene. Hybridization of the cloned cDNAs to genomic DNA identifies a single-copy gene, and using somatic cell hybrid analysis, we have mapped the corresponding gene locus (designated TNNT2) to the long arm of chromosome 1 (1cen-qter).

p53 protein detected by immunohistochemical staining is not always mutant.

The expression of the tumour suppressor gene p53 was analyzed in a variety of human solid tumours by immunohistochemistry and direct DNA sequencing. Positive nuclear staining using a panel of anti-p53 antibodies was used to select tumours for further genetic analysis. Using PCR amplification followed by immobilization onto magnetic beads and direct sequencing, we sequenced exons 5-9 of the p53 gene from 9 melanomas, 8 nasopharyngeal carcinomas, 16 sporadic breast carcinomas and 11 patients from familial breast cancer families. No sequence alterations of the p53 gene were detected in either the melanoma or nasopharyngeal tumours and only 19% of the primary breast carcinomas showed a variant band indicative of a mutation. Our results indicate firstly that p53 mutations are not generally involved in the tumour types studied and secondly the data emphasize the disparity encountered when attempting to correlate p53 immunohistochemical positivity with mutations within the p53 gene.

Some oral carcinomas from Sri Lankan betel/tobacco chewers overexpress p53 oncoprotein but lack mutations in exons 5-9.

We reported a low prevalence (11%) of p53 expression detected by immunohistochemistry in oral squamous cell carcinomas associated with betel/tobacco chewing Sri Lankans (23). Five neoplasms which over-expressed p53 protein were used in the present study of mutations. Despite extensive sequence analysis no mutations were detected in exons 5 through 9 of the p53 gene in all the DNA samples of these neoplasms. Thus, over-expression of p53 is not necessarily synonymous with mutations in the exons studied, although mutations of this gene have been reported in oral and head and neck cancer from Japan and USA. The absence of mutations in betel/tobacco related carcinomas in this population may derive from differences in aetiology, carcinogen metabolism and susceptibility, DNA repair mechanisms and/or genetic predisposition.

Isolation of cDNA clones for a 50 kDa glycoprotein of the human erythrocyte membrane associated with Rh (rhesus) blood-group antigen expression.

The Rh blood-group antigens are associated with human erythrocyte membrane proteins of approx. 30 kDa (the Rh30 polypeptides). Heterogeneously glycosylated membrane proteins of 50 and 45 kDa (the Rh50 glycoproteins) are coprecipitated with the Rh30 polypeptides on immunoprecipitation with anti-Rh-specific mono- and poly-clonal antibodies. We have isolated cDNA clones representing a member of the Rh50 glycoprotein family (the Rh50A glycoprotein). We used PCR with degenerate primers based on the N-terminal amino acid sequence of the Rh50 glycoproteins and human genomic DNA as a template and cloned and sequenced three types of PCR product of the expected size. Two of these products, Rh50A and Rh50B, gave the same translated amino acid sequence which corresponded to the expected Rh50 glycoprotein sequence but had only 75% DNA sequence similarity. The third product (Rh50C) contained a single base deletion, and the translated amino acid sequence contained an in-frame stop codon. We have isolated cDNA clones containing the full coding sequence of the Rh50A glycoprotein. This sequence predicts that it is a 409-amino acid N-glycosylated membrane protein with up to 12 transmembrane domains. The Rh50A glycoprotein shows clear similarity to the Rh30A protein in both amino acid sequence and predicted topology. Our results are consistent with the Rh30 and Rh50 groups of proteins being different subunits of an oligomeric complex which is likely to have a transport or channel function in the erythrocyte membrane. We mapped the Rh50A gene to human chromosome 6p21-qter, showing that genetic differences in the Rh30 rather than the Rh50 genes specify the major polymorphic forms of the Rh antigens.

Abnormal expression of wild type p53 protein in normal cells of a cancer family patient.

Mutations in the p53 gene are the commonest specific genetic change in human cancer. In normal tissues, p53 protein is present in such low quantities that it is not readily detectable by immunochemical techniques. However, in many tumour cells large amounts of p53 protein accumulate and can be seen by simple immunohistochemical staining; this is generally attributed to the accumulation of stabilised, mutant protein. We have found a mother and daughter, who both have a history of breast cancer, who show strong immunohistochemical staining of p53 in most of their normal epithelial and mesenchymal cells. Their family has a history of multiple cancers developing at an early age. Detailed protein analysis and gene sequencing of material obtained from cultured cells, grown from a skin biopsy taken from the daughter, suggest that her cells contained large quantities of normal (unmutated) p53. We suggest that this phenotype defines a new inherited cancer susceptibility syndrome that is distinct from the germ-line mutations in p53 found in some Li-Fraumeni families. This new syndrome affects p53 tumour suppressor function through an indirect mechanism that stabilises normal p53. It remains to be established whether this mechanism also contributes to the accumulation of p53 in sporadic cancers.

Assignment of the chromosomal locus of the human 30-kDal Rh (rhesus) blood group-antigen-related protein (Rh30A) to chromosome region 1p36.13----p34.

Using a panel of somatic cell hybrids, we have localised the 30-kDal Rhesus blood group-antigen-related protein to human chromosome 1 in the region p36.13----p34. This confirms the localisation of this protein described previously using cytogenetic and linkage analyses.

The human cardiac troponin I locus: assignment to chromosome 19p13.2-19q13.2.

The three major troponin I isoforms are encoded by separate genes and are expressed in a muscle-type-specific manner. A human cardiac troponin I cDNA has recently been isolated and used to establish the genomic location of the cardiac troponin I gene locus (designated TNNC1). By somatic cell hybrid analysis, the locus for TNNC1 maps to human chromosome 19 and can be localised to the region p13.2-q13.2.

Growth hormone regulates expression of rat liver cytochrome P-450 15 beta at a pretranslational level.

Polyclonal antibodies to rat liver microsomal cytochrome P-450 15 beta-hydroxylase were used to screen a female rat liver cDNA expression library. Recombinant plasmid C-6, isolated from a positive colony, contained a cDNA of 1000 base pairs which hybridized to a single, 1.7 kilobase class of mRNA on Northern blot analysis. Semi-quantitative slot blots showed that the level of this mRNA was 17-fold lower in male rat liver than in females. Continuous infusion of human growth hormone to male rats caused them to express female levels of C-6 mRNA, while the same daily dose of hormone given intermittently had no effect. A high degree of correlation of the levels of C-6 mRNA with 15 beta-hydroxylase protein levels and catalytic activities was found, demonstrating that the sexually differentiated, growth hormone-dependent expression of the 15 beta-hydroxylase is regulated at a step preceding translation.

Hypothalamo-pituitary regulation of cytochrome P-450(15) beta apoprotein levels in rat liver.

The regulation of the sexually differentiated steroid sulfate 15 beta-hydroxylase, cytochrome P-450(15) beta of female rat liver has been investigated. Specific antibodies raised to isozyme P-450(15) beta were used with the Western blot technique to quantitate the specific levels of P-450(15) beta in liver microsomes. The method demonstrated that the levels of the protein are about 16-fold higher in female than in male microsomes and also showed that the specific microsomal content of P-450(15) beta is controlled by GH. Hypophysectomy of female animals resulted in a decrease of P-450(15) beta to male levels. Continuous infusion of human GH, mimicking the female pattern of GH secretion in intact male animals, caused an elevation of the P-450(15) beta level to that of the female. The same dose of human GH in hypophysectomized male or female animals raised the P-450(15) beta level 8-fold or 50% of that seen in normal females. Infusion of ovine PRL to intact male rats had no effect on P-450(15) beta levels, whereas infusion of rat GH caused a 4-fold increase. Thus, the regulation of P-450(15) beta by GH is mainly associated with the somatogenic properties of the hormone. Furthermore, sc injection of rat GH every 12 h, mimicking the male pattern of GH secretion, had no effect on P-450(15) beta levels, demonstrating the importance of the GH secretory pattern in regulation of the specific protein levels. Postpubertal castration of male animals did not influence the microsomal P-450(15) beta content, whereas neonatal castration led to a feminization of the P-450(15) beta content in the adult male rat. Administration of estradiol valerate to male animals caused complete feminization of P-450(15) beta levels, whereas administration of androgen to female animals caused a decrease to male levels. Before 21 days of age, the P-450(15) beta level was slightly higher in male than in female rats. At 35 days, however, the P-450(15) beta level in female rats had increased almost 100-fold, whereas the levels in males increased only slightly. These changes are concomitant with the development of the sexual differentiation of the GH secretory pattern, supporting the role of GH in P-450(15) beta regulation. In conclusion, isozyme P-450(15) beta is a GH-regulated enzyme specific for female rats. The low level of the protein in males is probably explained by neonatal androgenic programming of the GH secretory pattern.

Hormonal and developmental regulation of expression of the hepatic microsomal steroid 16 alpha-hydroxylase cytochrome P-450 apoprotein in the rat.

The hormonal regulation of the sexually differentiated cytochrome P-450 isozyme which catalyzes 16 alpha-hydroxylation of testosterone and 4-androstene-3,17-dione in male rat liver (P-450(16) alpha) was investigated. Estradiol valerate injection of male rats caused a decrease in P-450(16) alpha levels to almost the female level, while methyltrienolone injection had the reverse effect in female animals. Hypophysectomy abolished the sex difference in P-450(16) alpha levels. Human growth hormone infusion into male rats, mimicking the female pattern of growth hormone secretion, caused a feminization of P-450(16) alpha levels. The same effect was also seen in hypophysectomized rats of both sexes. In contrast, a different administration schedule involving 12 h injections of human growth hormone, mimicking the male pattern of growth hormone secretion, caused a masculinization of P-450(16) alpha levels in hypophysectomized rats, at a daily dose which causes feminization when given by infusion. Thus, the level of expression of P-450(16) alpha in the liver is dependent on the temporal pattern of blood growth hormone levels. While infusion of rat growth hormone into male rats also feminized the P-450(16) alpha levels, infusion of ovine prolactin had no effect. Ontogenic studies showed that the developmental pattern of P-450(16) alpha expression in the liver coincided with the known pattern of development of the sexual differentiation of hepatic steroid 16 alpha-hydroxylase activity and of the diurnal pattern of growth hormone secretion.

Sexual differentiation of cytochrome P-450 in rat liver. Evidence for a constitutive isozyme as the male-specific 16 alpha-hydroxylase.

Cytochrome P-450 isozyme RLM5 [Cheng, K-C., and J.B. Schenkman, J. Biol. Chem. 257:2378-2385 (1982)], possessing a high steroid 16 alpha-hydroxylating activity, was isolated from male rat livers, and the hypothesis that this protein is the sexually differentiated 16 alpha-hydroxylase was tested. Isozyme RLM5 was not detected when the same purification procedure was applied to female rat liver microsomes. However, a female isozyme named DEa and possessing a similar Mr was obtained from similar column fractions. The DEa isozyme had insignificant steroid-hydroxylating activity and a substrate specificity different from isozyme RLM5. The two proteins could also be distinguished in their primary structures and immunochemical properties. Rabbit antibodies were raised to isozyme RLM5 and were made specific by immunoabsorption with a crude female cytochrome P-450 fraction coupled to Affi-Gel 10. The antibodies were used in a Western blot immunoassay to demonstrate that isozyme RLM5 can be detected in liver microsomes of male rats at levels at least 20 times higher than those in the female, and that its sexual differentiation is neonatally imprinted by androgen. The antibodies were able to specifically inhibit 70% of the testosterone 16 alpha-hydroxylase activity in male rat liver microsomes but had no effect on the activity in females. It was concluded that isozyme RLM5 is the major sexually differentiated microsomal 16 alpha-hydroxylase.

Purification, characterization, and pituitary regulation of the sex-specific cytochrome P-450 15 beta-hydroxylase from liver microsomes of untreated female rats.

A sex-differentiated form of cytochrome P-450 has been purified to electrophoretic homogeneity from liver microsomes of untreated female rats. The purified preparation contained 17 nmol of P-450/mg of protein and had a minimum molecular weight of 50,000. The final preparation was active in the hydroxylation of 5 alpha-[3H]androstane-3 alpha, 17 beta-diol 3,17-disulfate in the 15 beta position, with a turnover number of 2.6 nmol/min/nmol P-450 and is designated "P-450 15 beta" on the basis of this activity. Cytochrome P-450 15 beta is isolated essentially in the low-spin state and has a CO-reduced difference spectral maximum at 449 nm. Comparison of the female protein with the corresponding P-450 fraction from male rats revealed an absence of the 15 beta band in the male electrophoretic profile. Specific antibodies to isozyme 15 beta were used with a Western blot technique to demonstrate the virtual absence of the protein in male microsomes. This method was also used to demonstrate that hypophysectomy of female rats resulted in undetectable levels of the 15 beta-hydroxylase, while continuous infusion of growth hormone to normal male animals increased the 15 beta-hydroxylase level to that of female. P-450 15 beta is proposed to be the enzyme responsible for the predominance of 15 beta-hydroxylated steroid sulfate metabolites in excreta of female rats, and their absence in males. The same purification procedure for female rat liver microsomes also yielded another purified cytochrome P-450 characterized by a minimum molecular weight of 52,500, termed P-450 DEa, which was inefficient in the 15 beta-hydroxylation of 5 alpha-[3H]androstane-3 alpha, 17 beta-diol 3, 17-disulfate. No evidence was obtained that this form is sexually differentiated.