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Background: In the Western Hemisphere, 90% of bladder cancers are transitional cell carcinomas, while only 7% are classified as squamous cell carcinomas. In contrast, in Egypt and regions of the Middle East and Africa, where infection by the trematode Schistosoma haematobium is endemic, squamous cell carcinoma is the most common bladder cancer as well as the most common cancer in men.Purpose: We planned experiments to understand the genetic defects underlying the development of squamous cell carcinoma and to determine if the morphologically and clinically distinct squamous cell carcinoma and transitional cell carcinoma of the bladder evolve following different genetic alterations.Methods: Squamous cell carcinoma specimens from high-risk (Egypt, n = 19) and low-risk (Sweden, n = 12) populations were examined for genetic defects known to be involved in transitional cell carcinoma tumorigenesis. Homozygous deletions of the CDKN2 tumor suppressor gene were detected by comparative multiplex polymerase chain reaction. Mutations in the CDKN2 and p53 (also known as TP53) genes were analyzed by single-strand conformation polymorphism and DNA sequencing. Immunohistochemical staining of p53 protein was also performed. Allelic losses in chromosome arms 9p, 9q, and 17p were determined by microsatellite analysis.Results: Homozygous deletions and sequence mutations in the CDKN2 gene were found in 67% (eight of 12) of squamous cell carcinoma specimens, a frequency three times higher than that reported for uncultured transitional cell carcinomas (P = .009). Hemizygous and homozygous deletions in 9p, where CDKN2 resides, were found in 92% (11 of 12) of uncultured squamous cell carcinomas, while only about 39% (35 of 90) of transitional cell carcinomas showed these losses (P = .001). Deletions in 9p with no change in 9q were found in 92% (10 of 11) of squamous cell carcinomas compared with only 10% (11 of 110) of transitional cell carcinomas (P < .001) reported in the literature. The frequency of p53 mutations in squamous cell carcinomas was similar to that reported for invasive transitional cell carcinomas (60%), but the type and position of mutations differed between the two tumor types. Allelic losses in chromosome arm 17p, where the p53 gene resides, were found to be less frequent in squamous cell carcinomas (38%) than in invasive transitional cell carcinomas (60%).Conclusions: Our results suggest that a putative tumor suppressor gene on 9p, possibly CDKN2, may contribute to squamous cell carcinoma tumorigenesis. Our data on squamous cell carcinoma and previously reported data on transitional cell carcinoma indicate that these two bladder carcinomas differ in their genetic alterations, suggesting that distinct underlying genetic defects may explain, at least in part, the pathological differences between the two tumors of the bladder epithelium.Implications: Development of diagnostic and therapeutic strategies for squamous cell carcinoma of the bladder based on its distinct genetic alterations is warranted. [ABSTRACT FROM AUTHOR]

The progression of cancers from primary tumors to invasive and metastatic stages accounts for the overwhelming majority of cancer deaths. Understanding the molecular events which promote metastasis is thus critical in the clinic. Translational control is emerging as an important factor in tumorigenesis. The messenger RNA (mRNA) cap-binding protein eIF4E is an oncoprotein that has an important role in cancer initiation and progression. eIF4E must be phosphorylated to promote tumor development. However, the role of eIF4E phosphorylation in metastasis is not known. Here, we show that mice in which eukaryotic translation initiation factor 4E (eIF4E) cannot be phosphorylated are resistant to lung metastases in a mammary tumor model, and that cells isolated from these mice exhibit impaired invasion. We also demonstrate that transforming growth factor-beta (TGFβ) induces eIF4E phosphorylation to promote the translation of Snail and Mmp-3 mRNAs, and the induction of epithelial-to-mesenchymal transition (EMT). Furthermore, we describe a new model wherein EMT induced by TGFβ requires translational activation via the non-canonical TGFβ signaling branch acting through eIF4E phosphorylation.

Cyclin-dependent kinase subunit (Cks) proteins are small cyclin-dependent kinase-interacting proteins that are frequently overexpressed in breast cancer, as well as in a broad spectrum of other human malignancies. However, the mechanistic link between Cks protein overexpression and oncogenesis is still unknown. In this work, we show that overexpression of Cks1 or Cks2 in human mammary epithelial and breast cancer-derived cells, as well as in other cell types, leads to override of the intra-S-phase checkpoint that blocks DNA replication in response to replication stress. Specifically, binding of Cks1 or Cks2 to cyclin-dependent kinase 2 confers partial resistance to the effects of inhibitory tyrosine phosphorylation mediated by the intra-S-phase checkpoint, allowing cells to continue replicating DNA even under conditions of replicative stress. Because many activated oncoproteins trigger a DNA damage checkpoint response, which serves as a barrier to proliferation and clonal expansion, Cks protein overexpression likely constitutes one mechanism whereby premalignant cells can circumvent this DNA damage response barrier, conferring a proliferative advantage under stress conditions, and therefore contributing to tumor development.

Cks proteins associate with cyclin-dependent kinases and have therefore been assumed to play a direct role in cell cycle regulation. Mammals have two paralogs, Cks1 and Cks2, and individually deleting the gene encoding either in the mouse has previously been shown not to impact viability. In this study we show that simultaneously disrupting CKS1 and CKS2 leads to embryonic lethality, with embryos dying at or before the morula stage after only two to four cell division cycles. RNA interference (RNAi)-mediated silencing of CKS genes in mouse embryonic fibroblasts (MEFs) or HeLa cells causes cessation of proliferation. In MEFs CKS silencing leads to cell cycle arrest in G(2), followed by rereplication and polyploidy. This phenotype can be attributed to impaired transcription of the CCNB1, CCNA2, and CDK1 genes, encoding cyclin B1, cyclin A, and Cdk1, respectively. Restoration of cyclin B1 expression rescues the cell cycle arrest phenotype conferred by RNAi-mediated Cks protein depletion. Consistent with a direct role in transcription, Cks2 is recruited to chromatin in general and to the promoter regions and open reading frames of genes requiring Cks function with a cell cycle periodicity that correlates with their transcription.

Deregulation of cyclin E, an activator of cyclin-dependent kinase 2 (Cdk2), has been associated with a broad spectrum of human malignancies. Yet the mechanism linking abnormal cyclin E expression to carcinogenesis is largely unknown. The gene encoding the F-box protein hCdc4, a key component of the molecular machinery that targets cyclin E for degradation, is frequently mutated in endometrial cancer, leading to deregulation of cyclin E expression. Here we show that hCDC4 gene mutation and hyperphosphorylation of cyclin E, a parameter that usually correlates with hCDC4 mutation, have a strong statistically significant association with polypoidy and aneuploidy in endometrial cancer. On the contrary, elevated expression of cyclin E by itself was not significantly correlated with polyploidy or aneuploidy when tumors of similar grade are evaluated. These data suggest that impairment of cell cycle regulated proteolysis of cyclin E may be linked to carcinogenesis by promoting genomic instability.

hCDC4, the gene that encodes the F-box protein responsible for targeting cyclin E for ubiquitin-mediated proteolysis, has been found to be mutated in a number of primary cancers and cancer-derived cell lines. We have observed that functional inactivation of hCDC4 does not necessarily correlate with elevated levels of cyclin E in tumors. Here we show, however, that hCDC4 mutation in primary tumors correlates strongly with loss of cell cycle regulation of cyclin E. Similarly, a breast carcinoma-derived cell line mutated for hCDC4 exhibits cell cycle deregulation of cyclin E, but periodic expression is restored by reintroducing hCDC4 via retroviral transduction. Conversely, small interfering RNA-mediated silencing of hCdc4 deregulates cyclin E with respect to the cell cycle. These results indicate that hCdc4 function is an absolute prerequisite for cell cycle regulation of cyclin E levels, and loss of hCdc4 function is sufficient to deregulate cyclin E.

We generated mice lacking Cks2, one of two mammalian homologs of the yeast Cdk1-binding proteins, Suc1 and Cks1, and found them to be viable but sterile in both sexes. Sterility is due to failure of both male and female germ cells to progress past the first meiotic metaphase. The chromosomal events up through the end of prophase I are normal in both CKS2-/- males and females, suggesting that the phenotype is due directly to failure to enter anaphase and not a consequence of a checkpoint-mediated metaphase I arrest.

Cyclin-dependent kinase 2 activated by cyclin E is involved in the initiation of DNA replication and other S phase functions. Consistent with this role, cyclin E protein accumulates at the G1-S phase transition and declines during early S phase. This profile of expression is the result of periodic transcription and ubiquitin-mediated proteolysis directed by SCF(hCdc4). However, in many types of human tumors cyclin E protein is elevated and deregulated relative to the cell cycle by an unknown mechanism. Here, we show that the F-box protein hCdc4 that targets cyclin E to the SCF (Skp1-Cull-F-box) protein ubiquitin ligase is mutated in at least 16% of human endometrial tumors. Mutations were found either in the substrate-binding domain of the protein or at the amino terminus, suggesting a critical role for the region of hCdc4 upstream of the F-box. hCDC4 gene mutations were accompanied by loss of heterozygosity and correlated with aggressive disease. The hCDC4 gene is localized to chromosome region 4q32, which is deleted in over 30% of human tumors. Our results show that the hCDC4 gene is mutated in primary human tumors and suggest that it may function as a tumor suppressor in the genesis of many human cancers.

The eukaryotic cell cycle consists of a series of sequential phases, the order of which is highly regulated to ensure the faithful transmission of intact genome equivalents to daughter cells. Progression through the cell cycle depends on the activity of cyclin-dependent kinases (Cdks), which drive the transitions between phases by targeting numerous, but largely unknown, substrates for phosphorylation. The activity of Cdks is subject to both positive and negative regulation by their temporal association with cyclins and Cdk inhibitors, respectively. Whereas Cdks are constitutively expressed throughout the cell cycle, the levels of cyclins and Cdk inhibitors are regulated by both transcriptional and post-transcriptional processes. The discovery that many cyclins and Cdk inhibitors are unstable proteins has implicated regulated protein degradation as a critical mechanism in cell cycle control. Proteolysis allows for the rapid removal of cell cycle regulators promoting irreversible transitions between cell cycle phases. The rapid removal of positive regulators prevents them from interfering with regulation of subsequent cell cycle events. In this review, we highlight the recent advances of our understanding of how a recently discovered ubiquitin ligase, designated SCF, contributes to mammalian cell cycle control.

Cyclin E, one of the activators of the cyclin-dependent kinase Cdk2, is expressed near the G1-S phase transition and is thought to be critical for the initiation of DNA replication and other S-phase functions. Accumulation of cyclin E at the G1-S boundary is achieved by periodic transcription coupled with regulated proteolysis linked to autophosphorylation of cyclin E. The proper timing and amplitude of cyclin E expression seem to be important, because elevated levels of cyclin E have been associated with a variety of malignancies and constitutive expression of cyclin E leads to genomic instability. Here we show that turnover of phosphorylated cyclin E depends on an SCF-type protein-ubiquitin ligase that contains the human homologue of yeast Cdc4, which is an F-box protein containing repeated sequences of WD40 (a unit containing about 40 residues with tryptophan (W) and aspartic acid (D) at defined positions). The gene encoding hCdc4 was found to be mutated in a cell line derived from breast cancer that expressed extremely high levels of cyclin E.

Cyclin E, a regulatory subunit of cyclin-dependent kinase 2 (Cdk2), is an important regulator of entry into S phase in the mammalian cell cycle. In normal dividing cells, cyclin E accumulates at the G1/S-phase boundary and is degraded as cells progress through S phase. However, in many human tumours cyclin E is overexpressed and the levels of protein and kinase activity are often deregulated relative to the cell cycle. It is not understood how alterations in expression of cyclin E contribute to tumorigenesis. Here we show that constitutive cyclin-E overexpression in both immortalized rat embryo fibroblasts and human breast epithelial cells results in chromosome instability (CIN). In contrast, analogous expression of cyclin D1 or A does not increase the frequency of CIN. Cyclin-E-expressing cells that exhibit CIN have normal centrosome numbers. However, constitutive overexpression of cyclin E impairs S-phase progression, indicating that aberrant regulation of this process may be responsible for the CIN observed. These results indicate that downregulation of cyclin-E/Cdk2 kinase activity following the G1/S-phase transition may be necessary for the maintenance of karyotypic stability.

Kaposi's sarcoma (KS) develops in a variety of clinical states and is the most common tumor seen in patients with HIV-1 infection. KS develops as a multifocal mucocutaneous disease with subsequent spread to visceral organs, and it has been argued to be a benign proliferation caused by its multifocality at initial presentation, lack of aneuploidy, and spontaneous regression upon withdrawal of immunosuppressive agents in iatrogenically induced disease. We wished to determine whether KS lesions are clonal, indicative of a true neoplasm. Also, we tested whether multifocal KS lesions are clonally related, derived from a common progenitor cell or of independent cellular origin. We studied the X-chromosome inactivation pattern of the human androgen receptor gene in tumor biopsies of women with KS. This procedure tests for the clonality of a tissue specimen, a hallmark of neoplasia. Each specimen was microdissected to minimize normal cell contamination. Of 12 evaluable cases, 10 were HIV-seropositive and 2 were HIV-seronegative. Twenty-four biopsies from the 12 patients were examined. Five cases were consistent with individual KS lesions being clonal. In two cases, multiple KS specimens derived from the individual patients had different androgen receptor alleles inactivated, proving unequivocally that these KS lesions arose independently from distinct transformed cells. In seven cases, only a polyclonal pattern of inactivation was observed, whereas two others had tumor areas of both clonal and polyclonal inactivation patterns. These findings suggest that KS can be a clonal neoplasm, and in some of the cases multiple KS lesions in a given patient can arise from independent cellular origins and acquire clonal characteristics. The polyclonal inactivation pattern observed in other KS lesions may represent a premalignant stage or false negative results.

Mutatins of the p53 tumor suppressor gene are rare in nasopharyngeal carcinoma (NPC) patients who reside in high-risk areas, such as Southeastern China. Among this high-risk group, a pre-existing infection with the EBV and consumption of Cantonese salted fish are closely associated with NPC. We investigated the prevalence of p53 mutations in 28 primary NPC specimens from white (including Hispanic) and African-American patients in Los Angeles, who are at low risk for NPC. Using PCR-based single-strand conformational polymorphism and direct sequencing, we found four mutations (14%) in exons 5-8 of the p53 gene in four patients. All were C-to-T transition mutations: two were present in exon 5-one at codon 142 [CCT (Pro)-->CTT (Leu)] and another at codon 144 [CAG (Gln)-->TAG (stop codon)]. The other two mutations were identified in exon 8: one at codon 273 [CGT (Arg)-->CAT (His)], a CpG site, and one at codon 271, a silent mutation [GAG (Glu)-->GAA (Glu)]. This is the first report investigating the presence of p53 missense mutations in NPC among a low-risk population. Our data indicate that p53 is also an infrequent event among NPC patients at low risk for the disease.

We have developed a simple and reproducible fingerprinting method for screening the genome for regions of DNA that have altered patterns of DNA methylation associated with oncogenic transformation. Restriction enzymes with different sensitivities to cytosine methylation in their recognition sites were used to digest genomic DNAs from primary tumors, cell lines, and normal tissues prior to arbitrarily primed PCR amplification. Fragments that showed differential methylation were cloned and sequenced after resolving the PCR products on high-resolution polyacrylamide gels. The cloned fragments were then used as probes for Southern analysis to confirm differential methylation of these regions in colon tissues and cell lines. Forty-four DNA fragments associated with a total of five different regions of genomic DNA containing methylation sites were detected in 10 matched sets of normal and tumor colon DNAs and 7 colon cancer cell lines. A novel CpG island was also isolated that was found to be frequently hypermethylated in bladder and colon tumors. We have demonstrated that this technique is a rapid and efficient method that can be used to screen for altered methylation patterns in genomic DNA and to isolate specific sequences associated with these changes.

The most common genetic alteration identified to date in bladder cancer is loss of heterozygosity (LOH) of chromosome 9, suggesting the presence of possible tumor suppressor genes on this chromosome. We attempted to map the location of these genes by analyzing 69 primary transitional cell carcinomas of the bladder with a panel of microsatellite markers for LOH on chromosome 9. Monosomy 9 (defined by LOH of all informative markers analyzed on 9p and 9q) was detected in 26 of 69 (38%) tumors, and 22 of 69 (32%) tumors showed subchromosomal deletions. Twelve tumors (17%) demonstrated partial LOH of chromosome 9 and indicated two distinct regions of LOH. Eight tumors showed distal allelic loss of 9q with a minimal region of common deletion flanked proximally by marker GSN on 9q33. Six tumors showed proximal allelic loss of 9p and 9q with a minimal area of common deletion flanked by markers D9S970 on 9p12 and D9S283 on 9q21. Two tumors showed loss of both the distal region of 9q and the proximal region of 9p and 9q, which were separated by a possible 6-44 cM of retained genetic material. The proximal minimal area of common deletion excluded 9q22.3-q31 to where two putative tumor suppressor genes, the nevoid basal cell carcinoma syndrome and multiple self-healing squamous epithelioma (ESS1) genes, have been mapped. The growth arrest-specific gene (GAS1), a candidate tumor suppressor gene, was included within the proximal minimal region. We evaluated the GAS1 gene for its potential role in bladder cancer using single-strand conformational polymorphism to screen for mutations in GAS1 in 10 bladder cancer cell lines and 14 primary bladder tumors. A polymorphism at codon 88 was noted in one primary bladder tumor, but no other abnormalities were found, suggesting that another potential tumor suppressor gene important to bladder cancer resides in these minimally deleted regions. Because the nevoid basal cell carcinoma syndrome gene has long been speculated to be a putative tumor suppressor gene in bladder cancer and this gene has recently been characterized as the human homologue of the Drosophila patched gene (PTC), 20 primary bladder tumors with chromosome 9q LOH were screened for mutations in PTC using single-strand conformational polymorphism and heteroduplex analysis. No alterations were found in any of the samples analyzed. Furthermore, 4 of 37 noninvasive papillary (Ta) tumors demonstrated loss of all 9q markers with retention of 9p, whereas no Ta tumor showed loss of 9p with retention of all 9q markers, suggesting that LOH of 9q is the earlier event in bladder tumorigenesis. In summary, our results indicate two tumor suppressor loci associated with proximal chromosome 9p to q and distal chromosome 9q that may be important in bladder cancer. GAS1 and PTC do not seem to be frequently mutated in bladder cancer.

Previous studies of chimeric animals and human tissues have shown the clonal nature of organ development, giving clues as to the normal development of organs and also to abnormal developments, such as atheromatous plaques. The clonal nature of bladder cancer in female patients has been demonstrated, but little has been known of the clonal development of the normal urothelium. Using an X chromosome inactivation analysis of cells microdissected from histologic slides from the female human bladder, macroscopic urothelial patches of monoclonality were detected. These patches are about 120 mm.2 in size, contain about 2 x 10(6) cells each and reflect the presence of coherent cellular families composed of stem cells and their differentiated derivatives. The large size of these patches was surprising when compared with previously reported patch sizes in other organ systems. The patches most probably are composed of the descendants of the original founder cells, which would suggest that only 200 to 300 cells participated in the formation of the urothelium. The limited number of stem cells, each giving rise to millions of cells may provide an explanation for the "field defect" that is often referred to in the pathogenesis of bladder cancer, as different cell patches may possess different predispositions to tumorigenesis.

Alterations in DNA methylation patterns are one of the earliest and most common events in tumorigenesis. Overall levels of genomic methylation often decrease during transformation, but localized regions of increased methylation have been observed in the same tumors. We have examined changes in the methylation status of the muscle determination gene myoD, which contains a CpG island, as a function of oncogenic transformation. This CpG island underwent de novo methylation during immortalization of 10T1/2 cells, and progressively more sites became methylated during the subsequent transformation of the cells to oncogenicity. The greatest increase in methylation occurred in the middle of the CpG island in exon 1 during transformation. Interestingly, no methylation was apparent in the putative promoter of myoD in either the 10T1/2 cell line or its transformed derivative. The large number of sites in the CpG island that became methylated during transformation was correlated with heterochromatinization of myoD as evidenced by a decreased sensitivity to cleavage of DNA in nuclei by MspI. A site in the putative promoter also became insensitive to MspI digestion in nuclei, suggesting that the chromatin structural changes extended beyond the areas of de novo methylation. Unlike Lyonized genes on the inactive X chromosome, whose timing of replication is shifted to late S phase, myoD replicated early in S phase in the transformed cell line. Methylation analysis of myoD in DNAs from several human tumors, which presumably do not express the gene, showed that hypermethylation also frequently occurs during carcinogenesis in vivo. Thus, the progressive increase in methylation of myoD during immortalization and transformation coinciding with a change in chromatin structure, as illustrated by the in vitro tumorigenic model, may represent a common mechanism in carcinogenesis for permanently silencing the expression of genes which can influence cell growth and differentiation.

An elevated risk of bladder cancer has been reported in the endemic region of 'black foot disease' on the southwest coast of Taiwan and may be related to high arsenic levels in artesian well water. Thirteen urothelial tumors from this endemic region were examined for mutations in exons 5-8 of the p53 gene to identify the effects of possible exogenous factors at the DNA level. DNA was extracted from archival tissue after microdissection of tumors and analyzed by PCR-SSCP (polymerase chain reaction-based single strand conformation polymorphism), followed by direct sequencing. Eight cases (62%) showed mutations and 9 of the 10 point mutations observed were transitions. The type and position of the mutations were not significantly different when compared with the spectra of p53 mutations previously reported for transitional cell carcinomas (TCCs). However, two of the mutations were CGC-->CAC base changes at codon 175, a mutational hotspot for many tumor types but previously unreported in TCCs except in cases associated with inflammatory agents. Three of the tumors examined were found to contain double mutations, a relatively rare mutagenic event in human cancers. Our results suggest that the agents responsible for the high risk of bladder cancer in the black foot disease region may operate through an inflammation-based mechanism which increases the amount of DNA damage per mutagenic event.

Noninvasive transitional cell carcinomas of the bladder can have two distinct morphologies suggesting they contain different genetic alterations. Papillary transitional cell carcinomas (T(a) tumors) are often multifocal and only occasionally progress, whereas flat tumors (carcinomas in situ, CIS), frequently progress to invasive disease. We examined 216 bladder tumors of various stages and histopathologies for two genetic alterations previously described to be of importance in bladder tumorigenesis. Loss of heterozygosity of chromosome 9 was observed in 24 of 70 (34%) T(a) tumors but was present in only 3 of 24 (12%) CIS and dysplasia lesions (P = 0.04). In contrast, only 1 of 36 (3%) T(a) tumors contained a p53 gene mutation compared to 15 of 23 (65%) CIS and dysplasias (P < 0.001), a frequency comparable to that observed in muscle invasive tumors (25 of 49; 51%). The presence of p53 mutations in CIS and dysplasia could explain their propensities to progress since these mutations are known to destabilize the genome. Analysis of several tumor pairs involving a CIS and an invasive cancer provided evidence that the chromosome 9 alteration may in some cases be involved in the progression of CIS to more invasive tumors, in addition to its role in the initiation of T(a) tumors. However, the CIS and secondary tumor were found to contain different genetic alterations in some patients suggesting divergent progression pathways. Bladder carcinogenesis may therefore proceed through two distinct genetic alteration pathways responsible for generating superficial tumors with differing morphologies and pathologies.

Somatic instability at microsatellite repeats was detected in 6 of 200 transitional cell carcinomas of the bladder. Instabilities were apparent as changes in (GT)n repeat lengths on human chromosome 9 for four tumors and as alterations in a (CAG)n repeat in the androgen receptor gene on the X chromosome for three tumors. Single locus alterations were detected in three tumors, while three other tumors revealed changes in two or more loci. In one tumor we found microsatellite instability in all five loci analyzed on chromosome 9. The alterations detected were either minor 2-base pair changes or larger (> 2 base pairs) alterations in repeat length. All six tumors were low stage (Ta-T1), suggesting that these alterations can occur early in bladder tumorigenesis.

Seventy-three transitional cell carcinomas of the bladder were analyzed by immunohistochemistry for p53 nuclear accumulation, and the results were compared to mutations detected in the p53 gene by single strand conformational polymorphism analysis (SSCP) and DNA sequence analysis. Immunohistochemical studies were performed on formalin-fixed, paraffin-embedded tissue sections. A highly significant association between the presence of p53 mutations and p53 nuclear reactivity as detected by immunohistochemistry was found (P = 0.0001). Of 32 tumors that demonstrated p53 mutations by SSCP, 27 (84%) showed p53 nuclear reactivity. Of the five cases that did not demonstrate p53 nuclear reactivity, four had mutations in exon 5. However, of 41 tumors with no evidence of p53 mutation by molecular analysis, 12 (29%) showed p53 immunoreactivity. This indicates that immunohistochemical methods may be more sensitive than SSCP in detecting p53 mutations or that discordant cases represent tumors with accumulation of wild type p53 protein, without mutations at the p53 locus. Of the 15 tumors that were found to have mutations at exon 8, 13 demonstrated high-intensity homogeneous p53 nuclear reactivity by immunohistochemistry, and all mutations located at codon 280 demonstrated high-intensity homogeneous immunoreactivity. However, three of three tumors with exon 6 mutations demonstrated low-level p53 immunoreactivity, and four of six tumors with mutations in exon 5 showed no detectable p53 nuclear reactivity. This indicates that the heterogeneity of immunoreactivity observed when analyzing p53 nuclear accumulation may be related to the site of the p53 gene mutation. Information on tumor grade, stage, lymph node status, disease-free interval, and overall survival were available in 54 patients who had undergone cystectomy. A significant association was observed between p53 alterations (detected by immunohistochemistry and SSCP) and histological tumor grade (P = 0.003) and stage (P = 0.01). We conclude that the immunohistochemical detection of p53 nuclear accumulation in formalin-fixed, paraffin-embedded tissue is highly associated with mutations in the p53 gene; this association has now been demonstrated in a large number of tumors. The heterogeneity of p53 nuclear reactivity seems to be related to the site of mutation in the p53 gene. A small proportion of tumors with a p53 gene mutation do not demonstrate immunohistochemically detectable p53 nuclear accumulation. Furthermore, a small but substantial proportion of tumors demonstrate p53 nuclear reactivity but do not show detectable mutations in the p53 gene by SSCP. Finally, both grade and stage of bladder cancer are related to p53 alterations, detected by immunohistochemistry or molecular methods.(ABSTRACT TRUNCATED AT 400 WORDS)

The tumors of 20 patients with multifocal primary transitional cell carcinoma of the bladder or lymph node metastases were examined for molecular genetic defects which we have previously found to be present in > 50% of invasive tumors. These included loss of heterozygosity (LOH) of chromosome 9, which occurs in superficial as well as invasive bladder tumors, and LOH of chromosome 17p and p53 mutations, which are commonly found only in invasive tumors. Analysis of multiple or recurrent primary tumors in 7 patients for these markers was generally consistent with recently published data that the tumors are monoclonal in origin and that p53 mutations occur as a late event in the generation of invasive bladder cancers. Comparison of the primary tumors and metastases to regional lymph nodes in 14 patients demonstrated a complete concordance between the molecular genetic defects present, showing that LOH of chromosomes 9 and 17p and p53 mutations occurred in the primary tumors before metastasis. Because of the importance of chromosome 9 in bladder cancer, we mapped the location of a putative tumor suppressor gene by restriction fragment length polymorphism analysis of 123 cases obtained in this and earlier studies. Most of the tumors showed LOH for more than one marker on chromosome 9. Results of mapping of 4 tumors with partial deletion of chromosome 9 suggests that the tumor suppressor gene is located between 9p12 and 9q34.1.

A distinct mutational spectrum for the p53 tumor suppressor gene in bladder carcinomas was established in patients with known exposures to cigarette smoke. Single-strand conformational polymorphism analysis of exons 5 through 8 of the p53 gene showed inactivating mutations in 16 of 40 (40%) bladder tumors from smokers and 13 of 40 (33%) tumors from lifetime nonsmokers. Overall, 13 of the 50 (26%) total point mutations discovered in this and previous work were G:C-->C:G transversions, a relatively rare mutational type in human tumors. In six tumors, identical AGA (Arg)-->ACA (Thr) point mutations at codon 280 were observed, suggesting a mutational hotspot in these tumors. Comparison of the mutational spectra from smokers and nonsmokers revealed no obvious differences in the types or positions of inactivating mutations; however, 5 of 15 tumors containing point mutations from cigarette smokers had double mutations, four of which were tandem mutations on the same allele. No double mutations were found in tumors from nonsmoking patients. None of the mutations in smokers were G:C-->T:A transversions, which would be anticipated for exposure to the suspected cigarette smoke carcinogen 4-aminobiphenyl. The results suggest that, although cigarette smoke exposure may not significantly alter the kinds of mutations sustained in the p53 gene, it may act to increase the extent of DNA damage per mutagenic event.

Alterations in the p53 tumor suppressor gene and Epstein-Barr virus status were investigated in 15 nasopharyngeal carcinoma (NPC) biopsies, 4 xenografts, and 2 cell lines from the Cantonese region of southern China. One other established NPC cell line obtained from a northern Chinese patient was also studied. Restriction fragment length polymorphism analysis revealed a loss of heterozygosity for chromosome 17p, where the p53 gene resides, in only one of 15 NPC biopsies. Polymerase chain reaction-single-stranded conformational polymorphism analysis and direct sequencing failed to detect sequence alterations in exons 5 through 8 of the p53 gene in the 15 tumors and in the 4 NPC xenografts, all of which tested positive for Epstein-Barr virus. In contrast, the 3 NPC cell lines were all negative for Epstein-Barr virus and contained G----C transversions in the p53 gene, with cell lines CNE-1 and CNE-2 harboring identical AGA (arginine) to ACA (threonine) changes at codon 280. These results suggest that p53 inactivation is not a necessary component of nasopharyngeal carcinogenesis in Cantonese but may be important in the establishment of cell lines derived from these tumors.

A method using selective ultraviolet radiation fractionation followed by polymerase chain reaction (PCR) can analyze specific cell subsets present on a microscope section. Direct ultraviolet radiation of fixed and stained tissue sections prevents subsequent amplification by PCR. An "umbrella" or dot placed physically over small numbers of pure cell populations selected by microscopic examination protects these cells from the ultraviolet inactivation. The DNA in these protected cells can be specifically amplified while no signal is derived from the unprotected surrounding cells. Specific amplification was demonstrated by detecting human papillomavirus sequences only if infected cells were protected. Similarly, loss of heterozygosity at the p53 locus was documented by selective dotting of normal or tumor cells. The method allows the specific and sensitive molecular genetic analysis of small numbers of cells histologically identified and selected under the microscope.

The fifth base in human DNA, 5-methylcytosine, is inherently mutagenic. This has led to marked changes in the distribution of the CpG methyl acceptor site and an 80% depletion in its frequency of occurrence in vertebrate DNA. The coding regions of many genes contain CpGs which are methylated in sperm and serve as hot spots for mutation in human genetic diseases. Fully 30-40% of all human germline point mutations are thought to be methylation induced even though the CpG dinucleotide is under-represented and efficient cellular repair systems exist. Importantly, tumor suppressor genes such as p53 also contain methylated CpGs and these serve as hot spots for mutations in some, but not all, human cancers. Comparison of the spectrum of mutations present in this gene in different human cancers allows for predictions to be made on the molecular mechanisms of tumorigenesis.

Approximately 4% of cytosine residues in human DNA are modified post-synthetically into 5-methylcytosine (5mC) which is the only modified base present in vertebrate DNA. The function of 5mC is not fully understood, but methylation of promoter regions is often associated with transcriptional inactivity and may be part of a gene silencing mechanism. While undermethylation of promoter regions is correlated with expression, the same does not seem to be true for the remainder of genes since many genes are expressed while containing 5mC in their coding regions. This is significant because 5mC is known to be inherently mutagenic and it has been suggested that it is responsible for 30-40% of all human germline point mutations. We have used direct genomic sequencing to examine the methylation status of CpG sequences which serve as potential methylation sites in the human p53 gene. These sites, which are known to be hotspots for mutations in several human cancers, were found to be methylated in the target human tissues examined. The results suggest that 5mC may play a substantial role as an endogenous mutagen in the p53 gene and that the generation of these mutations does not require the direct interaction of a carcinogen with DNA. We have also compared the spectrum of p53 mutations reported in the literature for various human tumors. The patterns of mutations seen in different tumor types vary considerably and 5mC contributes to 63% of point mutations in colorectal cancer but only 13% in lung cancer. Mutations in lung cancer are therefore caused by a different mechanism than colorectal cancer and this presumably requires the direct interaction of carcinogens with DNA. Assessment of the proportion of 5mC induced mutations in the p53 gene therefore allows for an estimate of the relative importance of endogenous and exogenous mechanisms of carcinogenesis.

Core pentapeptides and an octapeptide (Peptide T) computer deduced from amino acid sequences from vasoactive intestinal peptide (VIP) and the 120 gp external envelope of the HIV (AIDS) virus and synthesized have been reported to have important in vitro and in vivo activity including inhibition of HIV binding to CD4 surface antigens of brain cells and lymphocytes and limitation of HIV infectivity. Two of these core pentapeptides, peptide TTNYT (Peptide T [4-8]) and peptide TDNYT (VIP [7-11]), are reported here, on acute infusion into cannulated afferent popliteal lymphatics of sheep, to produce prompt and marked depressions in the output of both small recirculating and blast lymphocytes into popliteal lymph node efferent lymph. As with a prior VIP infusion study, there appeared to be a selective effect on T4 (CD4) lymphocytes, with a marked predominance of T4 (CD4) lymphocytes in the lymphocyte depleted efferent lymph.

Induced systemic arterial hypotension by intravenous nitroprusside administration and by acute arterial occlusion in sheep have been found to reduce lymphocyte traffic as mirrored in the output of lymphocytes into the efferent lymph of peripheral lymph nodes. In the present series of experiments in sheep with chronically cannulated efferent lymphatics of peripheral lymph nodes, induced and monitored systemic arterial hypertension with intravenous pump infusions of phenylephrine or dopamine both produced sharp increases in the output of lymphocytes into efferent lymph in all of 27 studies. The increases in lymphocyte output with dopamine were more sustained and less associated with evidence of lymphoid tissue damage than with phenylephrine. Phenylephrine infusions were attended by a high incidence of gross bleeding into the efferent lymph, of increased coagulability of efferent lymph in the absence of gross bleeding and of prolonged depression of lymphocyte outputs after the cessation of intravenous infusion.

Vasoactive intestinal peptide (VIP) is a 28 amino acid-residue neurovascular and gut peptide with a number of important biological activities. Recent in vitro studies suggest an immunomodulatory (depressant) role for VIP. In the present in vivo studies, employing the Hall and Morris sheep lymphocyte traffic model, acute infusions of VIP into cannulated afferent lymphatics of popliteal lymph nodes produced prompt and marked depressions in the output of both small recirculating and blast lymphocytes into popliteal efferent lymph, with a selective effect on T4 (CD4) lymphocytes. It has been suggested that the HIV (AIDS) virus may employ VIP or VIP-like receptors on brain cells and lymphocytes for intracellular access.

In the past decade, the main interest in the involvement of prostaglandin E2 (PGE2) in the immune response has been concerned with its role in immunomodulation (suppression) both in vitro and in vivo. Comparatively little attention has been devoted to its immunostimulatory role. It has been suggested that PGE2, like histamine, may function as a 'double agent', initially triggering, facilitating and augmenting a stimulatory immune response and later modulating, limiting and contributing to the turning off of this response. We here report an early (within minutes) immunostimulatory involvement of PGE2 (and thromboxane A2) in the sheep, with prompt elevations in levels of PGE2 and thromboxane B2 in popliteal lymph node efferent lymph following drainage area immunization with killed Salmonella muenchen bacteria. These elevations were associated with an increase in efferent lymph flow and an equally prompt but limited depression of lymphocyte outputs into efferent lymph ('shutdown', 'recruitment'). Local increases in blood flow and vascular permeability probably play important roles in these events.

General anaesthesia of sheep with ketamine and xylazine has been found to produce a profound and prolonged depression in lymphocyte traffic through primary peripheral lymph nodes, as mirrored in the output of lymphocytes into efferent lymph. In this study, the depression has been found to be associated with a marked and sustained elevation in prostaglandin E2 (PGE2) levels in efferent lymph. The degree and duration of lymphocyte output depression was found to be modulated (diminished), both in degree and duration, by study-node drainage-area stimulation from prior surgery, inflammation or bacterial immunization. Even when the anaesthesia-associated lymphocyte-output depression was modulated by drainage-area inflammation, the period of lymphocyte-output depression was correlated still with elevated levels of PGE2 in efferent lymph. When drainage-area stimulation was produced by bacterial immunization (killed Salmonella muenchen), the anaesthesia-associated depression in lymphocyte output into efferent lymph (small as well as blast) was accompanied by a depression in antibody output into efferent lymph.

Substance P, an 11 amino acid residue vasoactive neurotransmitter peptide, has been found on acute infusion (50 micrograms) into cannulated afferent lymphatics of popliteal lymph nodes of sheep to produce marked elevations in both efferent lymph flow and in the outputs of both blast and small recirculating lymphocytes into popliteal node efferent lymph (chronically cannulated). These elevations were characterized by a delay in the onset of major elevations, a marked prolongation of the elevations and a substantially greater stimulative effect on the output of blast lymphocytes. It is suggested that the number and types of substance P receptors on lymphocytes and in sheep peripheral lymph nodes may be responsible for these observations. Infusion of substance P, known for involvement in pain impulse transmission, was able to briefly overcome anaesthesia-induced depression in lymphocyte traffic. The substance P-induced alterations in lymph flow and lymphocyte traffic in vivo were demonstrated to be due to local rather than systemic effects of substance P.