Coming into focus: the nonovarian origins of ovarian cancer.

BACKGROUND: The traditional view of epithelial ovarian cancer asserts that all tumor subtypes share a common origin in the ovarian surface epithelium (OSE) DESIGN: A literature review was carried out to summarize the emerging understanding of extraovarian sources of epithelial ovarian carcinomas. RESULTS: Historically, there were no diagnostic criteria for documenting the origin of ovarian epithelial carcinomas. Moreover, there are no normal epithelial tissues in the ovary with morphologic similarities to these tumors. In fact, no precursor lesions have ever been reproducibly identified in the ovary. However, there is a strong correlation between extrauterine Müllerian tissue and the development of ovarian carcinomas, tumors of low malignant potential, and cystadenomas. The most recent support for this hypothesis comes from the careful analysis of risk-reducing bilateral salpingo-oopherectomy specimens from BRCA1 or BRCA2 mutation carriers. These studies showed that a significant majority of high-grade serous ovarian carcinomas, the most common subtype, arise from the fallopian tube fimbriae rather than the OSE. CONCLUSIONS: Mounting evidence indicates that the vast majority of epithelial ovarian carcinomas are not ovarian in origin. Extrauterine Müllerian epithelium from various sites in the reproductive tract likely accounts for the diverse morphology and behavior of these tumors.

BRCA genes: lessons learned from experimental and clinical cancer.

Advances in the study of BRCA1 and BRCA2 gene functions have relied on the development of animal models for seeking to explore further what we have learned from the human disease. Specifically, mouse models of a 'triple-negative' breast cancer (utilizing conditional knockout of BRCA1 and p53 in the breast), of an endometrioid ovarian cancer (based on oncogenic kras and loss of function of pten), and of anatomic and functional consequences of BRCA1 mutations in granulosa cells, have led to further inquiry into the pathogenesis and therapeutic consequences of genetic alterations. A striking susceptibility of these murine malignancies to platinum drugs has emerged, providing further confidence in their relevance to the human disease. In addition to these models, the pathogenesis of high-grade serous disease derived from risk-reducing surgeries in mutation carriers has pointed to a role of mutations in p53 commonly encountered in tubal intraepithelial carcinomas.

Quantitative analysis of associations between DNA hypermethylation, hypomethylation, and DNMT RNA levels in ovarian tumors.

How hypermethylation and hypomethylation of different parts of the genome in cancer are related to each other and to DNA methyltransferase (DNMT) gene expression is ill defined. We used ovarian epithelial tumors of different malignant potential to look for associations between 5'-gene region or promoter hypermethylation, satellite, or global DNA hypomethylation, and RNA levels for ten DNMT isoforms. In the quantitative MethyLight assay, six of the 55 examined gene loci (LTB4R, MTHFR, CDH13, PGR, CDH1, and IGSF4) were significantly hypermethylated relative to the degree of malignancy (after adjustment for multiple comparisons; P < 0.001). Importantly, hypermethylation of these genes was associated with degree of malignancy independently of the association of satellite or global DNA hypomethylation with degree of malignancy. Cancer-related increases in methylation of only two studied genes, LTB4R and MTHFR, which were appreciably methylated even in control tissues, were associated with DNMT1 RNA levels. Cancer-linked satellite DNA hypomethylation was independent of RNA levels for all DNMT3B isoforms, despite the ICF syndrome-linked DNMT3B deficiency causing juxtacentromeric satellite DNA hypomethylation. Our results suggest that there is not a simple association of gene hypermethylation in cancer with altered DNMT RNA levels, and that this hypermethylation is neither the result nor the cause of satellite and global DNA hypomethylation.

Human decidua is a major source of renin.

Plasma prorenin levels are elevated in normal pregnant women. Current evidence suggests renin production by tissues of the uteroplacental unit contribute to this elevation. The purpose of this investigation was to define the source of renin biosynthesis within the human uteroplacental unit and to characterize the renin produced. RNA extraction and Northern blot analysis consistently demonstrated renin mRNA expression in uterine lining both in the pregnant (decidua) and nonpregnant states (endometrium) and in fetal chorion laeve, which is inseparable from the decidua. In contrast, renin mRNA expression was not detected in basal plate and intertwin chorion (which is separate from decidua), amnion, myometrium, or placental villi. The total renin content in decidual homogenates was two- to threefold greater than in endometrial homogenates, and cultured human decidual cells produced significantly more total renin than cultured human endometrial cells, suggesting that pregnancy enhanced renin production by the cells lining the uterus. Immunoblot analysis and [3H]leucine incorporation identified 47,000-mol wt prorenin as the major form of renin produced by cultured human decidual cells. These studies indicate that maternal decidua is the major source of prorenin in the uteroplacental unit.

Telomerase activity in benign and malignant epithelial ovarian tumors.

BACKGROUND: Ovarian epithelial tumors include benign lesions lacking invasive and metastatic abilities (cystadenomas) in addition to malignant lesions (carcinomas). An intermediate category, called tumors of low malignant potential (LMP), is also recognized. The merit of this classification is being challenged because the clinical behavior of LMP tumors appears closer to that of cystadenomas than to that of carcinomas. PURPOSE: To verify our hypothesis that the expression of the enzyme telomerase distinguishes these two categories of ovarian epithelial tumors, we examined and compared such expression in ovarian cystadenomas and carcinomas. By examining the expression of telomerase in LMP tumors, we then sought to determine if these tumors were more closely related to cystadenomas or to carcinomas with regard to telomerase expression. METHODS: We examined a total of 64 consecutive ovarian tumors subdivided into 20 carcinomas, 17 LMP tumors, and 27 cystadenomas. We subsequently discarded three of the 27 cystadenomas because of the presence of admixed normal ovarian stroma in those specimens. Tumor subtyping was done without knowledge of the telomerase results, and telomerase assays were likewise interpreted without knowledge of tumor types. Telomerase activity was determined by use of the TRAP (i.e., telomeric repeat amplification protocol) assay. Differences between the proportions of tumors expressing this enzyme in each subgroup were evaluated by use of Fisher's exact test (two-sided). RESULTS: Telomerase activity was detected in all 20 carcinomas and in all 17 LMP tumors examined. In contrast, it was not detected in 19 of the 24 cystadenomas. These differences between rates of telomerase expression in either carcinomas or LMP tumors and those in cystadenomas were statistically significant (P<.0001). All five of the telomerase-positive cystadenomas belonged to a variant called papillary cystadenomas, whereas none of the telomerase-negative cystadenomas belonged to this variant (P<.0001). CONCLUSIONS AND IMPLICATIONS: The presence of telomerase expression in ovarian LMP tumors supports the merit of continuing to separate these tumors from cystadenomas, in spite of their apparent benign clinical course. The finding of telomerase expression in papillary cystadenomas suggests that such tumors may be mechanistically related to LMP tumors and should perhaps be reclassified as variants of LMP tumors. Lack of telomerase expression in ovarian cystadenomas raises questions about the alleged immortality of these tumors because expression of this enzyme is thought to be essential for continuous growth in adult tumors.

Detection of ovarian cancer cells: comparison of a telomerase assay and cytologic examination.

BACKGROUND: Telomerase is an enzyme essential for the normal replication of chromosomes. Telomerase activity is absent in most somatic cells in adults, but it is usually expressed in cancer cells, including ovarian carcinoma cells. Our principal goal was to compare the sensitivity of a telomerase assay, i.e., the telomeric repeat amplification protocol (TRAP) assay, with that of cytologic examination in detecting cancer cells in the peritoneal cavity of patients with ovarian carcinoma. METHODS: TRAP assays and cytologic examinations were performed on peritoneal washings and ascitic fluids from 42 patients with active ovarian carcinoma. Control specimens included washings from 29 patients with benign ovarian diseases and ascitic fluids from 14 patients with liver failure. We also evaluated the stability of telomerase in ascitic fluids left unprocessed at room temperature as well as the ability of the TRAP assay to detect cancer cells in mixtures containing large numbers of normal cells. RESULTS: Specimens from 37 (88%) of the 42 patients with ovarian carcinoma tested positive for telomerase. Cytologic examination detected cancer cells in only 27 of the telomerase-positive specimens (i.e., in specimens from 64% of the 42 patients). This difference of 24% (95% confidence interval = 17%-30%) in sensitivity between the two tests was statistically significant (two-sided P = .002). Specimens from five of the patients with ovarian carcinoma were cytologically negative and telomerase negative. All 43 control specimens were cytologically negative, but the TRAP assay detected telomerase in two of them. Telomerase activity was detected in unprocessed samples left at room temperature for 5 days and in mixtures containing a small number of cancer cells and a 2000- to 10000-fold excess of normal cells. CONCLUSIONS: Assaying for telomerase is more sensitive than cytologic examination in detecting cancer cells in the peritoneal cavity of patients with ovarian carcinoma.

Genetic disparity between morphologically benign cysts contiguous to ovarian carcinomas and solitary cystadenomas.

BACKGROUND: Ovarian carcinomas occasionally contain large, histologically benign cysts contiguous to the clearly malignant areas (cystadenocarcinomas). The question of whether such cysts are remnants of pre-existing benign tumors (cystadenomas) or constitute integral components of the carcinomas is important in clarifying the role of cystadenomas in ovarian carcinogenesis. It is also important for our general understanding of tumor heterogeneity, a phenomenon thought to result from the gradual accumulation of genetic abnormalities in initially homogeneous tumors. This question is also pertinent to the clinical management of ovarian cystadenomas, which are frequent in women of childbearing age and are usually treated surgically based on the possibility that they may give rise to carcinomas. PURPOSE: Reasoning that molecular markers of ovarian malignancy would be confined to the histologically malignant portions of cystadenocarcinomas if the morphologically benign portions are in fact pre-existing typical cystadenomas, we sought to verify that mutations in the p53 tumor suppressor gene are markers of malignancy in ovarian tumors and to determine the distribution of such mutations in cystadenocarcinomas. METHODS: We used immunohistochemical and DNA-sequencing techniques to analyze 46 ovarian carcinomas, 21 ovarian tumors of low malignant potential, and 16 solitary cystadenomas for the presence of p53 mutations. We then used similar techniques to examine the distribution of such mutations in different portions of cystadenocarcinomas. The observed differences in mutation frequencies were analyzed by the two-tailed Fisher's exact test. RESULTS: Mutations in the p53 gene were present in 24 (52%) of the 46 carcinomas, but they were absent in the 21 tumors of low malignant potential (P < .0001) and the 16 solitary cystadenomas (P = .0002). Six of six cystadenocarcinomas with p53 mutations showed the presence of the same mutations in the adjacent, histologically benign cysts. The mutations were seen not only in cells immediately adjacent to the carcinomas, but also throughout the morphologically benign cysts. Twenty (83%) of the 24 cases showing mutation of one p53 allele also showed loss of genetic heterozygosity, suggesting that the other p53 allele was deleted. Such allelic loss, if present in morphologically malignant portions of cystadenocarcinomas, was also observed in the contiguous cysts. CONCLUSIONS: Ovarian carcinomas can be distinguished from ovarian cystadenomas and tumors of low malignant potential by p53 mutations. The fact that the mutations were present in histologically benign cysts contiguous to ovarian carcinomas suggests that such cysts are not typical cystadenomas and may carry a genetic predisposition to carcinogenesis that is not present in ordinary cystadenomas.

Potential role of the inactivated X chromosome in ovarian epithelial tumor development.

BACKGROUND: Ovarian epithelial tumors can be divided into subcategories often regarded as different stages of neoplastic transformation. Cystadenomas belong to the least aggressive subgroup and are noninvasive and nonmetastatic. Ovarian tumors of low malignant potential (LMP) are intermediate between cystadenomas and carcinomas and show markedly reduced invasive and metastatic abilities. Invasion and metastasis are the hallmarks of carcinomas, which constitute the most aggressive subgroup and can be further subdivided into different grades. PURPOSE: We performed comparative allelotype analyses of ovarian cystadenomas, LMP tumors, and carcinomas, reasoning that such analyses could provide clues about the molecular determinants of their phenotypic differences. Because we realized that allelic losses involving the X chromosome might be associated with LMP tumor development, we determined whether such losses were interstitial and whether they involved the active or the inactive X chromosome. METHODS: Frequencies of loss of heterozygosity (LOH) at specific loci in every chromosomal arm were determined in 16 ovarian cystadenomas, 23 ovarian LMP tumors, 15 low-grade ovarian carcinomas, and 35 high-grade ovarian carcinomas by use of either the polymerase chain reaction (PCR) or Southern blot analyses. We took advantage of the fact that DNA methylation is an important mechanism of X-chromosome inactivation to determine whether losses involving the X chromosome were in the active or the inactive copy. We analyzed the methylation status of retained alleles on the X chromosome by determining whether they could be amplified by PCR after digestion with the methylation-sensitive restriction endonuclease Hpa II. RESULTS: High-grade carcinomas contained frequent(>50%) LOH in four autosomal chromosome arms, i.e., 6q, 13q, 17p, and 17q. Except for 13q, these same chromosomal arms showed frequent LOH in low-grade carcinomas. LOH in autosomal chromosomes was comparatively rare in LMP tumors and was absent in cystadenomas. In contrast, half (eight of 16) of LMP tumors informative for a locus in the proximal portion of chromosome Xq showed LOH at that locus. These losses were the result of interstitial deletions in six of the eight cases and involved the inactive copy of the X chromosome exclusively. Similar losses in the X chromosome were not seen in either cystadenomas or low-grade carcinomas. CONCLUSIONS AND IMPLICATIONS: LOH at multiple loci is associated with the development of ovarian carcinomas but not with the development of cystadenomas and LMP tumors. However, the integrity of a locus in chromosome Xq that possibly escapes X-chromosome inactivation is important for the control of LMP tumor development. The fact that this locus does not appear to be involved in the genesis of low-grade carcinomas suggests that LMP tumors are not precursors of such carcinomas.

Growth of normal and neoplastic urothelium and response to epidermal growth factor in a defined serum-free medium.

We have developed a defined serum-free medium for the in vitro growth of normal and neoplastic urothelium. Growth of epithelial cells in this medium was stimulated while proliferation of fibroblasts was inhibited. The medium allowed for the isolation of a new line from a high grade human bladder carcinoma. Several strains of normal human urothelium that could be passaged several times in culture were also obtained. Although growth of normal and neoplastic urothelial cells in the defined medium was not stimulated by epidermal growth factor, the presence of 50 ng/ml epidermal growth factor induced morphological changes suggestive of terminal maturation of normal urothelium, implying that epidermal growth factor either acted as an epithelial maturation factor or induced the secretion of such a factor.

A new in vivo model to study invasion and metastasis of human bladder carcinoma.

An animal model to investigate the invasive and metastatic properties of human bladder transitional cell carcinoma (HTCC) was established. Two long-term HTCC cell lines (RT4 and EJ) and one HTCC cell line derived in our laboratory (LD-71) were tumorigenic when injected s.c. into nude mice but had little potential to invade locally or metastasize before the animals succumbed to tumor burden. Experimental lung metastases were, however, observed in approximately 60% of animals given injections of RT4 or EJ cell lines in the tail vein. The cells were also implanted transurethrally into the urinary bladders of athymic mice. RT4 cells, which were originally isolated from a superficial papillary tumor, produced histologically noninvasive tumors after transurethral inoculation with no evidence of metastasis. In contrast EJ cells, which were originally isolated from a more aggressive tumor, produced invasive tumors in nude mouse bladders and metastasized to the lungs spontaneously. The invasive cell line LD-71 was weakly tumorigenic and was successfully implanted into the bladder on only one of five attempts. The human origin of the implanted tumors was documented by Southern blot analysis using human repetitive DNA as a probe. The results indicate that the site of injection strongly influences the behavior of the resulting tumors and that intravesical implantation of these HTCC cell lines produces pathological expression of invasiveness and metastatic potential.

Differential regulation of plasminogen activators by epidermal growth factor in normal and neoplastic human urothelium.

The regulation of urokinase (u-PA) and tissue plasminogen activator (t-PA) in cultured normal and neoplastic urothelium was examined because plasminogen activators (PAs) are thought to be important in malignancy. Both activators were synthesized by normal urothelial cells grown in vitro under chemically defined conditions. The level of t-PA activity decreased when normal urothelial cells reached saturation density, but was stimulated more than 10-fold by the addition of epidermal growth factor (EGF) to the culture medium. Northern blot analysis showed that the regulation occurred at the transcriptional level. On the other hand u-PA activity was regulated to a lower degree by EGF and was not affected by cell density. Immunohistochemical examination of urothelial cells in histology specimens showed that t-PA was present only in the apical cells facing the lumen, suggesting that the expression of the activity might be a marker for end-stage differentiation in vivo. In contrast to normal cells, tumor cell lines made only u-PA under all conditions tested, and the levels of its expression were either unaffected or slightly decreased by EGF. Tumor cells and normal cells therefore showed substantial differences in protease regulation by EGF.

Rapid degradation of extracellular matrix proteins by normal human uroepithelial cells.

The degradation of subendothelial and smooth muscle matrices by normal and neoplastic uroepithelial cells grown under serum-free conditions was examined. Normal urothelial cells were compared with neoplastic cells derived from a low grade papillary tumor (RT4) and a more invasive carcinoma (EJ). Low levels of degradation were observed with all cell types in serum-free medium alone. Supplementing the medium with plasminogen increased the degradative activity of each cell type. Logarithmically growing normal urothelial cells degraded extracellular matrix proteins 6 to 14 times faster on a per cell basis than their transformed counterparts. Analysis of the residual matrix constituents revealed that, while the levels of glycoprotein breakdown by the normal and neoplastic cells were similar, the normal cells degraded more of the collagen components than the neoplastic cells. Epidermal growth factor and cell density were examined as possible regulators of degradative activity. The neoplastic cells were not responsive to cell density as a regulatory factor and were only slightly responsive to epidermal growth factor. However, epidermal growth factor increased the degradative activity of logarithmically growing normal urothelial cells in the presence of plasminogen and the activity of confluent cells was increased to an even greater extent. Gelatin substrate gel analysis confirmed that the normal urothelial cells elaborated a more diverse set of gelatinases than the tumorigenic cells. Although normal urothelial cells had higher degradative abilities than their malignant counterparts, it is significant that the neoplastic cells were less responsive to regulatory signals in our model system. Thus, loss of regulatory mechanisms for protease secretion and matrix degradation may be a more important determinant of invasive ability in vivo than protease secretion or matrix degradation in vitro.

Southern blot analysis of DNA extracted from formalin-fixed pathology specimens.

We have developed a method for the extraction of DNA from formalin-fixed, paraffin-embedded pathology specimens. High-molecular-weight DNA was recovered from well-fixed nonautolyzed samples of viable tissue. DNA recovered from samples exposed to picric acid or mercuric chloride containing fixatives was not intact. Increasing the formalin fixation time decreased the amount of intact DNA available. When these limitations were taken into consideration, the procedure allowed for the removal of degraded and chemically modified DNA from the preparation, and the final product was suitable for quantitative and qualitative analysis by Southern or dot blotting techniques. Digestion with methylation-sensitive restriction endonucleases showed that DNA methylation patterns were not altered after formalin fixation.

Histologically benign or low-grade malignant tumors adjacent to high-grade ovarian carcinomas contain molecular characteristics of high-grade carcinomas.

It is presently not clear if ovarian carcinomas arise de novo or from benign precursors (cystadenomas) and if high-grade malignant tumors (carcinomas) develop from preexisting low-grade carcinomas. The presence of allelic losses on chromosome 11p15.5 distinguishes high-grade ovarian carcinomas from either low-grade carcinomas or cystadenomas. We therefore examined the distribution of such losses in different parts of heterogeneous tumors showing mixed histological grades or showing adjacent large histologically benign neoplasms. The results showed that all neoplastic areas, including those that were histologically benign or compatible with low-grade carcinomas, contained allelic losses at the above locus. This suggests that the morphologically less aggressive portions of these heterogeneous tumors were not typical cystadenomas or low-grade carcinomas and contained molecular abnormalities indicative of at least a predisposition to the high-grade carcinoma phenotype.

Distinction of low grade from high grade human ovarian carcinomas on the basis of losses of heterozygosity on chromosomes 3, 6, and 11 and HER-2/neu gene amplification.

We examined the frequencies of loss of heterozygosity at 13 different loci distributed on 9 chromosomes in 30 human ovarian carcinomas. The same tumors were also examined for the presence of amplification of the HER-2/neu and H-ras protooncogenes. The results confirmed earlier findings that losses of heterozygosity occurred at nonrandom frequencies on chromosomes 3, 6, and 11 in these tumors. None of the tumors examined showed amplification at the H-ras locus. The HER-2/neu gene, however, was amplified in approximately one-third of the tumors, in agreement with earlier studies from other laboratories. We subdivided our tumor specimens according to their histological grades, which can be regarded as representing different stages of tumor progression. Losses of heterozygosity on chromosomes 3 or 11 were not seen in low grade lesions, although they were present in most of the high grade tumors examined. Losses of heterozygosity on chromosome 6 as well as HER-2/neu amplification, in contrast, were present in several low grade tumors and were not more frequent in high grade lesions. We conclude that the latter two abnormalities are associated with cellular functions involved at earlier stages of ovarian tumor development, whereas inactivation of genes on chromosome 3 or 11 is associated with later steps that may be incompatible with the well differentiated phenotype.

Loss of heterozygosity on chromosome 13 is common only in the biologically more aggressive subtypes of ovarian epithelial tumors and is associated with normal retinoblastoma gene expression.

We examined the frequencies of loss of heterozygosity on chromosome 13 in 77 primary ovarian epithelial tumors subdivided into cystadenomas, tumors of low malignant potential, low grade carcinomas, and high grade carcinomas. Such losses were found in approximately 50% of high grade carcinomas but were not detected in any of the other tumor subtypes (P < 0.0001), suggesting a strong association between these abnormalities and the high grade carcinoma phenotype. The tumors were also examined for abnormalities in expression of the retinoblastoma susceptibility gene (RB). This was assessed by immunohistochemical staining of archival tumor sections with a polyclonal antibody directed against both the phosphorylated and the underphosphorylated forms of the RB protein. Most of the tumors, including those with allelic deletions on chromosome 13, showed normal RB nuclear protein staining patterns. We conclude that loss of RB expression is not a frequent event in primary ovarian carcinomas and that this gene is probably not the target of the frequent allelic deletions on chromosome 13 found in high grade ovarian carcinomas.

Loss of heterozygosity on chromosomal segments 3p, 6q and 11p in human ovarian carcinomas.

The genetic events involved in human ovarian carcinoma development are still largely unknown. We have used DNA recombinant technologies to examine the genome of normal and neoplastic tissues from 12 different patients with such tumors. We used cloned DNA sequences homologous to loci showing restriction fragment length polymorphisms on chromosomal segments 3p, 5p, 6q, 11p, or 21q to determine the frequencies of losses of constitutional heterozygosity at these loci in tumor DNAs. Losses affecting loci on 3p, 6q, and 11p were found in a high percentage of the cases examined. In contrast, losses of heterozygosity were not found when we used DNA probes for the other 2 above chromosomal segments. Thus, genetic losses involving chromosomal segments 3p, 6q and 11p occurred at non-random frequencies in ovarian carcinomas, suggesting that inactivation of genes located on these chromosomes played a role in their development.

Suppression of tumorigenicity in human ovarian cancer cell lines is controlled by a 2 cM fragment in chromosomal region 6q24-q25.

Multiple distinct regions of chromosome 6 are frequently affected by losses of heterozygosity in primary human ovarian carcinomas. We introduced a normal human chromosome 6 into HEY and SKOV-3 ovarian carcinoma cell lines using microcell-mediated chromosome transfer techniques to further investigate the role of this chromosome in ovarian tumorigenesis. The exogenous chromosome was stably propagated in the recipient cells based on fluorescence in situ hybridization (FISH) analyses with a chromosome 6 painting probe. The tumorigenicity of HEY and SKOV-3 cells was completely suppressed after transfer of chromosome 6, but not after transfer of a chromosome 11q13-qter fragment used as control. Using 46 polymorphic microsatellite markers, the region bounded by D6S1649 and D6S1564 was found to be commonly deleted in HEY: chromosome 6 tumorigenic revertant clones. The boundaries of the commonly deleted region could be further narrowed down to a 2 cM (based on the Whitehead genetic map) or 0.36 megabase (based on gdb mapping data) region between D6S1637 and D6S1564 after transferring the exogenous chromosome from revertants into mouse L cells and performing allelic deletion mapping studies against this mouse background. We conclude that this region contains a tumor suppressor gene important for the control of ovarian tumor development.

The mechanism of replication of phi X 174. XVIII. Gene A and A* proteins of phi X 174 bind tightly to phi X 174 replicative form DNA.

Evidence is presented that the gene A and A * proteins of bacteriophage phi X 174 form covalent associations with the 5' ends of the DNA molecules when superhelical phi X replicative form DNA is nicked by a combination of these proteins in vitro. This evidence is: 1, The 5' ends of the DNA molecules nicked by the gene A protein and reacted with bacterial alkaline phosphatase were protected against subsequent phosphorylation by polynucleotide kinase even after treatment of the nicked DNA with SDS and pronase followed by centrifugation on a high-salt neutral sucrose gradient. 2, Iodinated pronase-sensitive material remained attached to the nicked replicative form DNA and could not be removed by exposure to SDS or 2 M NaCl, either by sedimentation through high-salt neutral sucrose gradients, or by CsCl equilibrium centrifugation. 3, Iodinated pronase-sensitive material was detected on DNA that had been nicked during the reaction, but not on unreacted DNA. 4, Electrophoresis of the iodinated pronase-sensitive, DNA-bound material in SDS-polyacrylamide gels after DNAse digestion revealed that it was composed almost entirely polypeptides with electrophoretic mobilities similar to those of the gene A and A * proteins. We speculate that the gene * protein may be essential for normal progeny single-stranded DNA synthesis in vivo.

Collection of buccal cell DNA in seventh-grade children using water and a toothbrush.

We developed a simple and effective method for collecting a large quantity of buccal cell DNA in school-based studies of seventh-grade and older children. Seventh-grade students at schools in Wuhan, China brushed each buccal surface with a soft toothbrush and then rinsed with 10 ml of water. We added 5 ml of 99% ethanol to preserve the sample. Among 1563 samples transported at room temperature over 1 week and then stored for 13-14 months at -70 degrees C before extraction, using a modified Gentra Puregene protocol, the median total DNA yield was 108 microg, range of 14 to 416 microg. We assayed every 20th sample (n = 77) for NAT2 by the PCR, and all samples gave a 1093-bp product. From the 1563 samples, we obtained a result for single nucleotide polymorphisms in the interleukin-13 gene (at +2044) by RFLP-PCR on 98.8% and in the promoter of the myeloperoxidase gene (at -463) by real-time PCR on 99.7%. A water-rinse method, that we used among 12th-grade students in Southern California, gave a lower total DNA yield than the toothbrush rinse (median of 17 microg) and a slightly reduced ability to generate a PCR product. However, 26 of 27 water-rinse samples gave a result for two genes, albumin and CYP1A1, using real-time PCR methods. We did not quantify human, versus bacterial, DNA in our samples. However, given the amounts of total DNA required for genotyping, a sample with the median yield of 108 microg should suffice for approximately 2160 genotypes by RFLP-PCR methods or five times as many by real-time PCR. We recommend the toothbrush-rinse method, combined with a modified Gentra Puregene DNA extraction protocol, for large-scale, in-person collections of buccal cell DNA in children. The method requires only inexpensive, readily available materials and produces a large quantity of high-quality DNA for PCR analyses.