Thyroid hormone protects hepatocytes from HBx-induced carcinogenesis by enhancing mitochondrial turnover.

Infection by hepatitis B virus (HBV) accounts for 50-80% of hepatocellular carcinoma (HCC) development worldwide, in which the HBV-encoded X protein (HBx) has critical role in the induction of carcinogenesis. Several studies have shown that thyroid hormone (TH) suppresses HCC development and protects hepatocytes from HBx-induced damage, thus it is of interest to examine whether TH can protect hepatocytes from HBx-induced carcinogenesis. By treating HBx- transgenic mice with or without TH, we confirmed the protective effects of TH on HBx-induced hepatocarcinogenesis, which was achieved via reduction of reactive oxygen species (ROS) inflicted DNA damage. We further found that TH induced biogenesis of mitochondria (MITO) and autophagy of HBx-targeted MITO simultaneously, consequently leading to suppression of HBx-promoted ROS and carcinogenesis. Using microarray data analysis, this protective effect of TH was found to be mediated via activation of PTEN-induced kinase 1 (PINK1) in hepatocytes. PINK1, in turn, activated and recruited Parkin, an E3 ligase, to ubiquitinate MITO-associated HBx protein and trigger selective mitophagy. The pathological significance of the TH/PINK1 pathway in liver protection was confirmed by the concomitant decrease in expression of both TR and PINK1 in matched HCC tumor tissues and negatively correlated with aggressive progression of cancer and poor prognosis. Our data indicate that TH/PINK1/Parkin pathway has a critical role in protecting hepatocytes from HBx-induced carcinogenesis. Notably, several liver-targeting therapeutic derivatives of TH facilitating prevention or therapy of steatosis have been identified. Furthermore, our proof-of-concept experiments suggest that application of T3 constitutes an effective novel therapeutic or preventive option for HCC. Thus, the utilization of the agonists of TRs could be the meaningful strategy in liver relative diseases, ranging from simple hepatic steatosis to HCC.

Thyroid hormone receptor represses miR-17 expression to enhance tumor metastasis in human hepatoma cells.

MicroRNAs (miRNAs) are thought to control tumor metastasis through direct interactions with target genes. Thyroid hormone (T3) and its receptor (TR) are involved in cell growth and cancer progression. However, the issue of whether miRNAs participate in T3/TR-mediated tumor migration is yet to be established. In the current study, we demonstrated that T3/TR negatively regulates mature miR-17 transcript expression, both in vitro and in vivo. Luciferase reporter and chromatin immunoprecipitation (ChIP) assays localized the regions responding to TR-mediated repression to positions -2234/-2000 of the miR-17 promoter sequence. Overexpression of miR-17 markedly inhibited cell migration and invasion in vitro and in vivo, mediated via suppression of matrix metalloproteinases (MMP)-3. Moreover, p-AKT expression was increased in miR-17-knockdown cells that led to enhanced cell invasion, which was blocked by LY294002. Notably, low miR-17 expression was evident in highly metastatic cells. The cell migration ability was increased by T3, but partially reduced upon miR-17 overexpression. Notably, TRα1 was frequently upregulated in hepatocellular carcinoma (HCC) samples and associated with low overall survival (P=0.023). miR-17 expression was significantly negatively associated with TRα1 (P=0.033) and MMP3 (P=0.043) in HCC specimens. Data from our study suggest that T3/TR, miR-17, p-AKT and MMP3 activities are interlinked in the regulation of cancer cell metastasis.

Thyroid hormone suppresses cell proliferation through endoglin-mediated promotion of p21 stability.

Hypothyroidism has been associated with significantly elevated risk for hepatocellular carcinoma (HCC), although the precise underlying mechanisms remain unknown at present. Thyroid hormone (T3) and its receptor (TR) are involved in metabolism and growth. Endoglin is a T3/TR candidate target gene identified from our previous studies. Here, we demonstrated that T3 positively regulates endoglin mRNA and protein levels, both in vitro and in vivo. The thyroid hormone response elements of endoglin were identified at positions -2114/-2004 and -2032/-1973 of the promoter region using the electrophoretic mobility shift assay and chromatin immunoprecipitation assay. Endoglin was downregulated in the subgroups of HCC patients and significantly associated with histology grade (negative association, P=0.001), and this expression level was significantly associated with TRα1 in these HCC patients. Our results clearly indicate that p21 is involved in T3-mediated suppression of cell proliferation. Knock down of endoglin expression in HCC cells facilitated p21 polyubiquitination and promoted cell proliferation in the presence of T3. The data collectively suggest that T3/TR signaling suppresses cell proliferation by upregulating endoglin, in turn, affecting p21 stability. The results indicate that endoglin has a suppressor role to inhibit cell proliferation in HCC cell lines.

Thyroid hormone receptors promote metastasis of human hepatoma cells via regulation of TRAIL.

Although accumulating evidence has confirmed the important roles of thyroid hormone (T(3)) and its receptors (TRs) in tumor progression, the specific functions of TRs in carcinogenesis remain unclear. In the present study, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) was directly upregulated by T(3) in TR-overexpressing hepatoma cell lines. TRAIL is an apoptotic inducer, but it can nonetheless trigger non-apoptotic signals favoring tumorigenesis in apoptosis-resistant cancer cells. We found that TR-overexpressing hepatoma cells treated with T(3) were apoptosis resistant, even when TRAIL was upregulated. This apoptotic resistance may be attributable to simultaneous upregulation of Bcl-xL by T(3), because (1) knockdown of T(3)-induced Bcl-xL expression suppressed T(3)-mediated protection against apoptosis, and (2) overexpression of Bcl-xL further protected hepatoma cells from TRAIL-induced apoptotic death, consequently leading to TRAIL-promoted metastasis of hepatoma cells. Moreover, T(3)-enhanced metastasis in vivo was repressed by the treatment of TRAIL-blocking antibody. Notably, TRAIL was highly expressed in a subset of hepatocellular carcinoma (HCC) patients, and this high-level expression was significantly correlated with that of TRs in these HCC tissues. Together, our findings provide evidence for the existence of a novel mechanistic link between increased TR and TRAIL levels in HCC. Thus, TRs induce TRAIL expression, and TRAIL thus synthesized acts in concert with simultaneously synthesized Bcl-xL to promote metastasis, but not apoptosis.

Overexpression of CXCL1 and its receptor CXCR2 promote tumor invasion in gastric cancer.

BACKGROUND: The chemokine (C-X-C motif) ligand 1 (CXCL1) and its receptor CXCR2 are associated with metastasis potential. Our studies were designed to clarify the CXCL1 and CXCR2 expression patterns and to explore their potential role in gastric cancer. DESIGN: The expression of CXCL1 was determined in primary gastric cancer specimens using quantitative PCR, immunohistochemistry, and western blotting. To investigate the functional significance of CXCL1 expression, a CXCL1 expression vector and short hairpin RNA targeting the CXCL1 or CXCR2 were transfected into gastric cancer cell lines to examine the biological outcomes of these cells. RESULTS: The expression of CXCL1 and CXCR2 was higher in gastric cancer tissues compared with adjacent noncancerous tissues. The upregulation of CXCL1 correlated significantly with tumor progression, advanced stage of gastric cancer patients, and was one of the independent prognostic factors for patient's survival. Furthermore, cancer cells expressing CXCL1 stably exhibited an increase in their migration and invasion ability, whereas CXCL1 or CXCR2 depletion significantly reduced the migration and invasion ability of each cell line. CONCLUSIONS: These results provide strong evidence that CXCL1 plays an important role in gastric cancer progression and migration and suggest that CXCL1 is a promising marker for the detection and prognosis of gastric cancer.

Cathepsin H regulated by the thyroid hormone receptors associate with tumor invasion in human hepatoma cells.

Thyroid hormone, 3, 3', 5-triiodo-L-thyronine (T(3)), mediates cell growth, development and differentiation by binding to its nuclear receptors (TRs). The role of TRs in cancer is still undefined. Notably, hyperthyroxinemia has been reported to influence the rate of colon cancer in an experimental model of carcinogenesis in rats. Previous microarray analysis revealed that cathepsin H (CTSH) is upregulated by T(3) in HepG2-TR cells. We verified that mRNA and protein expression of CTSH are induced by T(3) in HepG2-TR cells and in thyroidectomized rats following administration of T(3). The possible thyroid hormone-responsive elements of the CTSH promoter localized to the nucleotides -2038 to -1966 and -1565 to -1501 regions. An in vitro functional assay showed that CTSH can increase metastasis. J7 cells overexpressing CTSH were inoculated into severe combined immune-deficient mice and these J7-CTSH mice displayed a greater metastatic potential than did J7-control mice. The clinicopathologic significance of CTSH expression in hepatocellular carcinoma (HCC) was also investigated. The CTSH overexpressing in HCC was associated with the presence of microvascular invasion (P=0.037). The microvascular invasion characteristic is closely related to our in vitro characterization of CTSH function. Our results show that T(3)-mediated upregulation of CTSH led to matrix metallopeptidase or extracellular signal-regulated kinase activation and increased cell migration. This study demonstrated that CTSH overexpression in a subset hepatoma may be TR dependent and suggests that this overexpression has an important role in hepatoma progression.

Increase of microRNA miR-31 level in plasma could be a potential marker of oral cancer.

BACKGROUNDS: Oral squamous cell carcinoma (OSCC) is a worldwide disease. MicroRNAs are endogenously expressed non-coding RNAs that have important biological and pathological functions. miR-31 was found markedly up-regulated in OSCC and several other malignancies. However, miR-31 expression was also down-regulated in the metastasis process of breast carcinoma. MATERIALS AND METHODS: Using quantitative RT-PCR analysis, we identified plasma miR-31 in OSCC patients (n = 43) and case controlled individuals (n = 21). Nine OSCC patients saliva were also analyzed. The Mann-Whitney test and Wilcoxon matched pairs test were used to compare the differences among the various clinical variants. RESULTS: miR-31 in plasma was significantly elevated in OSCC patients relative to age and sex-matched control individuals. This marker yielded a receiver operating characteristic curve area of 0.82 and an accuracy of 0.72 defined by leave-one-out cross-validation. In addition, the plasma miR-31 in patients was remarkably reduced after tumor resection suggesting that this marker is tumor associated. Our preliminary analysis also demonstrated the feasibility of detecting the increase of miR-31 in patient's saliva. CONCLUSION: This study concluded that plasma miR-31 could be validated a marker of OSCC for diagnostic uses.

Effects of tumor necrosis factor alpha on host immune response in chronic persistent tuberculosis: possible role for limiting pathology.

Reactivation of latent tuberculosis contributes significantly to the incidence of disease caused by Mycobacterium tuberculosis. The mechanisms involved in the containment of latent tuberculosis are poorly understood. Using the low-dose model of persistent murine tuberculosis in conjunction with MP6-XT22, a monoclonal antibody that functionally neutralizes tumor necrosis factor alpha (TNF-alpha), we examined the effects of TNF-alpha on the immunological response of the host in both persistent and reactivated tuberculous infections. The results confirm an essential role for TNF-alpha in the containment of persistent tuberculosis. TNF-alpha neutralization resulted in fatal reactivation of persistent tuberculosis characterized by a moderately increased tissue bacillary burden and severe pulmonic histopathological deterioration that was associated with changes indicative of squamous metaplasia and fluid accumulation in the alveolar space. Analysis of pulmonic gene and protein expression of mice in the low-dose model revealed that nitric oxide synthase was attenuated during MP6-XT22-induced reactivation, but was not totally suppressed. Interleukin-12p40 and gamma interferon gene expression in TNF-alpha-neutralized mice was similar to that in control mice. In contrast, interleukin-10 expression was augmented in the TNF-alpha-neutralized mice. In summary, results of this study suggest that TNF-alpha plays an essential role in preventing reactivation of persistent tuberculosis, modulates the pulmonic expression of specific immunologic factors, and limits the pathological response of the host.

Common metastatic carcinoma of California sea lions (Zalophus californianus): evidence of genital origin and association with novel gammaherpesvirus.

Tissues from 10 adult California sea lions (Zalophus californianus, seven females and three males) that had metastatic carcinoma in sublumbar area lymph nodes were examined histologically. A distinctive epithelial proliferative lesion interpreted as intraepithelial neoplasia was found in genital tracts of all ten animals; in vagina (5/7), cervix (7/7), uterus (3/7), penis (3/3) and prepuce (3/3). Intraepithelial neoplasia closely resembled metastatic carcinomas and was directly contiguous with invasive carcinoma in one animal. Rare eosinophilic intranuclear inclusion bodies were found in penile and preputial intraepithelial neoplasia (one animal), cervical intraepithelial neoplasia (one animal), invasive cervical carcinoma (one animal) and metastatic carcinoma (two animals). Electron microscopic examination of tissues from two sea lions (one with intraepithelial neoplasia and one with metastatic carcinoma) demonstrated viral particles consistent with a herpesvirus. An immunohistochemical stain for the latent membrane protein of Epstein-Barr virus was positive in intraepithelial neoplasia in one sea lion. Herpesvirus DNA sequences were detected by consensus primer polymerase chain reaction (PCR) in metastatic carcinomas from all four sea lions from which unfixed tumor samples were available. Results of sequencing were consistent with a novel gammaherpesvirus in the genus Rhadinovirus. DNA extracted from the four metastatic carcinomas also was tested for papillomavirus by Southern blot and PCR with consensus papillomavirus primers; all samples were negative by both methods. These findings support the genital origin of the sea lion carcinoma and implicate a novel gammaherpesvirus as a possible cause.

Intergenic complementation truncation mutants of cyclin-dependent kinase.

The Saccharomyces cerevisiae genes PHO80 and PHO85 encode, respectively, a cyclin and cyclin-dependent kinase, which negatively regulate PHO5 gene transcription by phosphorylating the transcription activator Pho4p. Cyclin-dependent kinases (CDKs) are highly conserved proteins, both within and between species. It was previously demonstrated, using reporter genes activated in yeast by Pho4p, that hybrid proteins in which over two-thirds of Pho85p were replaced with the homologous region from human Cdk2 retained the function of native Pho85p with respect to promoter repression. In the present study, various truncated forms of the hybrid human-yeast CDKs were tested for function. Surprisingly, truncations in which significant portions of the C-terminal region of the 291-residue hybrid CDK were deleted retained activity. Genes encoding human Cdk2 proteins which terminated after amino acids 151, 140, 130, 120 and 90 each complement a chromosomal pho85 gene disruption in which the HIS3 gene is inserted at codon 49. Truncated Cdk2 proteins containing less than 60 amino acids failed to complement the pho85::HIS3 gene disruption. Although the functional C-terminal truncations disrupt the ATP-binding and active sites of Cdk2, reporter gene repression mediated by these truncated proteins is apparently due to phosphorylation of Pho4p, since a gene in which the essential lysine codon at position 33 was converted to an arginine codon does not complement the chromosomal gene disruption. The human Cdk2 truncations were demonstrated to function through intergenic complementation. The intact Cdk2-Pho85 hybrid CDK complemented the pho85 mutation in yeast strains in which the entire PHO85 coding region was deleted from chromosome XVI. The C-terminal Cdk2 truncations, however, were non-functional in these strains and thus dependent for activity on the pho85 coding region which remained in the mutant pho85::HIS3 chromosomal locus. These genetic results are consistent with a model involving protein fragment complementation in which the active site of the CDK is bisected.

Genetic heterogeneity in ductal carcinoma of the breast.

Genetic heterogeneity in breast cancer has been observed both by cytogenetic and loss of heterozygosity (LOH) analyses; however, the frequency with which genetically heterogeneous clones arise is unknown. In this study, a panel of 115 breast carcinomas was analyzed to determine the extent of clonal divergence in tumor foci at progressive stages of tumor evolution. Intraductal, infiltrating, and metastatic tumor components were microdissected from each tumor and tested for LOH at 20 microsatellite markers on seven chromosomal arms. Of these cases, 24 (21%) demonstrated genetically divergent clones during tumor progression. Clonal divergence, inferred from discordant LOH patterns, was observed most commonly between intraductal and infiltrating tumor (18 cases), but was also demonstrated between infiltrating and metastatic tumor (11 cases). Discordant LOH was observed with markers on one chromosomal arm in 16 cases, on two in 7 cases, and on four in 1 case, and was observed most commonly with markers on 17p, 17q, and 16q. More detailed microdissection of four cases provided evidence for a specific chronology of genetic alterations occurring during the progression of each tumor. The results indicate that the different tumor components observed microscopically in breast cancer specimens often represent genetically divergent clones.

Molecular genetic evidence of a novel morbillivirus in a long-finned pilot whale (Globicephalus melas).

A long-finned pilot whale with morbilliviral disease was stranded in New Jersey. An immunohistochemical stain demonstrated morbilliviral antigen. Reverse transcriptase-polymerase chain reaction for morbillivirus P and N genes was positive. Novel sequences most closely related to, but distinct from, those of dolphin and porpoise morbilliviruses suggest that this virus may represent a third member of the cetacean morbillivirus group.

Loss of heterozygosity on chromosome 11p15 during histological progression in microdissected ductal carcinoma of the breast.

Microdissection of histologically identifiable components from formalin-fixed, paraffin-embedded tissue sections allows molecular genetic analyses to be correlated directly with pathological findings. In this study, we have characterized loss of heterozygosity (LOH) at chromosome 11p15 at different stages of progression in microdissected tumor components from 115 ductal carcinomas of the breast. Microdissected foci of intraductal, infiltrating, and metastatic tumors were analyzed to determine the stage of progression at which LOH at 11p15 occurs. LOH was detected in 43 (37%) of 115 cases. Foci of intraductal carcinoma could be microdissected from 85 cases, of which 30 (35%) showed LOH at some stage of progression. LOH was detected in the intraductal component in 26 of these 30 cases. Interstitial deletions were characterized by using a panel of 10 highly polymorphic markers. The smallest region of overlap (SRO) for LOH at 11p15 was bounded by the markers D11S4046 and D11S1758. LOH at 11p15.5 showed no correlation with estrogen receptor status, the presence of positive lymph nodes, tumor size, histological grade, or long-term survival. We conclude that 11p15 LOH usually occurs early in breast cancer development but less frequently does not develop until the infiltrating or metastatic stages of tumor progression.

Transactivation of the human MDR1 gene by hepatitis B virus X gene product.

BACKGROUND/AIMS: Persistent hepatitis B virus (HBV) infection may cause hepatocellular carcinoma. Patients with hepatocellular carcinoma are characterized by nonresponsiveness to chemotherapeutic agents. While many studies have been devoted to understanding the hepatocarcinogenesis mechanism of HBV, the possible relationship between HBV and the drug sensitivity phenotype of cancer cells has rarely been addressed. The hepatitis B virus X gene encodes a transcription transactivator which has been suggested to be a potential factor in viral hepatocarcinogenesis. The role of HBV pX in mediating the drug resistance phenotype of hepatoma cell lines was examined in this study. METHODS: Standard transfection and chloramphenicol acetyltransferase assay were utilized to examine the effect of HBV pX transactivator on a reporter gene under the control of the human multidrug resistance (MDR) 1 upstream regulatory elements. Selected Hep G2 clones with or without HBV pX expression were tested for their sensitivity towards various anti-cancer agents by utilization of MTT assay. RESULTS: The expression of HBV pX in both Hep G2 (p53+) and Hep 3B (p53-) cells resulted in transactivation of the reporter gene under control of the human MDR1 upstream regulatory elements. Northern blot analysis indicated that expression of the endogenous MDR1 gene was also elevated in Hep G2 clones with HBV pX expression. Decreased drug sensitivity towards adriamycin, vinblastine, and VP-16 was observed in Hep G2 clones with HBV pX expression. CONCLUSIONS: HBV pX can transactivate the MDR1 gene. Drug sensitivity was altered in Hep G2 cells with HBV pX expression.

Histologic, immunohistochemical, and polymerase chain reaction studies of bottlenose dolphins from the 1987-1988 United States Atlantic coast epizootic.

Tissues from 95 bottlenose dolphins (Tursiops truncatus) that died during the 1987-1988 US Atlantic coast epizootic and 11 bottlenose dolphins that died along the Atlantic coast prior to 1987 were examined histologically and immunohistochemically. Polymerase chain reaction (PCR) testing was performed on 36 of the epizootic and all of the pre-1987 cases. Epizootic cases had syncytia and rare intranuclear and intracytoplasmic inclusion bodies within lung, lymph node, and spleen. Lymphoid depletion was present in lymph node, spleen, and gut-associated lymphoid tissue of epizootic cases. Pre-1987 cases did not have these pulmonary and lymphoid lesions. A larger percentage of epizootic than pre-1987 cases had bacterial and/or fungal infections (primarily pneumonias), pulmonary and lymphoid tissue histiocytosis, mucocutaneous ulcers, and evidence of negative energy balance. Immunohistochemically, 49/95 (52%) epizootic dolphins were positive for morbilliviral antigen. Morbilliviral antigen was detected in lung, lymph node, spleen, thymus, skin, tongue, esophagus, liver, pancreas, gastrointestinal tract, urinary bladder, oviduct, and mammary gland by immunohistochemistry. PCR testing identified morbilliviral RNA in 35/36 (97%) epizootic cases tested. Neither morbilliviral antigen nor morbilliviral RNA were detected in pre-1987 cases. Histologic, immunohistochemical, and PCR results provide strong evidence that morbillivirus infection was the primary cause of the 1987-1988 bottlenose dolphin epizootic.

Changes of adenosine triphosphate-dependent calcium uptake in microsomal fractions of rat liver during sepsis.

BACKGROUND: Intracellular calcium concentration is an important regulator of cellular metabolism. Endoplasmic reticulum membranes play an important role in the regulation of cytoplasmic calcium in the mammalian liver. The characterization of the changes of calcium uptake in endoplasmic reticulum may contribute to the potential intracellular mechanisms for cellular dysfunction during sepsis. METHODS: The effects of sepsis on the calcium uptake in rough endoplasmic reticulum of rat liver were studied. Sepsis was induced by means of cecal ligation and puncture (CLP). The control rats underwent sham operation. Microsomal fractions were isolated from the liver with differential centrifugation. RESULTS: The calcium uptake by liver endoplasmic reticulum was decreased by 30% to 35% (p < 0.05) during early sepsis (9 hours after CLP) and by 38% to 43% (p < 0.05) during late sepsis (18 hours after CLP), respectively. The maximum velocity values for adenosine triphosphate (ATP) and for Ca2+ were also decreased by 25% to 37% (p < 0.05) during early sepsis and by 35% to 42% (p < 0.05) during late sepsis. The Michaelis-Menten constant for ATP and Ca2+ transport had no difference among three groups. The magnesium stimulation and vanadate inhibitory activity were also decreased by 17% to 38% (p < 0.05) during early sepsis and by 34% to 50% (p < 0.05) during late sepsis. CONCLUSIONS: These data demonstrate that ATP-dependent calcium uptake in rough endoplasmic reticulum of rat liver was impaired during early and late sepsis. Because the low intracellular calcium concentration plays an important role in the regulation of cellular function, an impairment in the ATP-dependent calcium uptake by endoplasmic reticulum during early and late sepsis may have a pathophysiologic significance in contributing to the development of altered hepatic metabolism during sepsis.

Differential expression of the human ST5 gene in HeLa-fibroblast hybrid cell lines mediated by YY1: evidence that YY1 plays a part in tumor suppression.

Through a mutational analysis of a differentially regulated enhancer, we present evidence that supports a role for the transcription factor YY1 in tumor suppression in HeLa/fibroblast somatic cell hybrids. The human ST5 gene was previously shown to be expressed as three RNA species, 4.6, 3.1 and 2.8 kb in length. Whereas the two larger species are expressed at similar levels in all cell lines examined, the 2.8 kb mRNA is expressed specifically in non-tumorigenic hybrids. In this study, the basis for the differential expression of this mRNA species was investigated. The message was shown to originate from a promoter located within an intron of the ST5 gene. An enhancer located approximaely 1500 nt upstream of the start site was required for cell type specific expression. Mutational analysis of this enhancer revealed an AP1 site and five YY1 sites which were necessary for full enhancer activity. Levels of YY1 DNA binding activity were found to be as much as 6-fold higher in the non-tumorigenic cells relative to the tumorigenic cells, while AP1 activity was similar in both cell types. These results suggest that a signaling pathway targeting YY1 may play an important role in tumor suppression in HeLa-fibroblast hybrids.

MDM2 amplification, P53 mutation, and accumulation of the P53 gene product in malignant fibrous histiocytoma.

Fifty-two cases of malignant fibrous histiocytoma (MFH) were evaluated for amplification of the MDM2 gene, mutation of the P53 gene, accumulation of the P53 gene product, and their relation to disease-free and overall survival. All tests were carried out on formalin-fixed, paraffin-embedded tissue samples. Amplification of the MDM2 gene was detected in 15 of 52 cases (29%). Six of 52 cases (12%) demonstrated abnormalities of the P53 gene. Sequence analysis detected point mutations in four cases and a 1-base pair deletion in one case, whereas differential polymerase chain reaction (dPCR) indicated that the P53 gene had been entirely deleted in one case. Eight of 52 cases (15%) demonstrated staining for the P53 protein in >10% of tumor cells. The presence of MDM2 amplification did not have a significant effect on either disease-free or overall survival. Patients with accumulation of the P53 gene product did not differ in disease-free or overall survival from patients without P53 accumulation. Survival also was not significantly different in patients with genetic aberration in P53. However, when the patients were stratified by histologic grade, the results indicated that patients with alterations in the P53 gene may have shorter overall survival.

Use of semiquantitative PCR to assess onset and treatment of Pneumocystis carinii infection in rat model.

The use of semiquantitative PCR (SQPCR) to assess Pneumocystis carinii pneumonia (PCP) infection and its response to treatment was studied with rats. Groups of eight rats were immunosuppressed with steroids for 3 to 12 weeks. Untreated controls were maintained for the same periods. Three groups of rats were treated with pentamidine, three groups were treated with trimethoprim-sulfamethoxazole, and three groups of rats were tapered from steroids. At various times during suppression, rats from the different groups were sacrificed. At necropsy, lungs were lavaged to obtain bronchoalveolar fluids and then homogenized. Bronchoalveolar fluids and homogenates were assayed by cyst counting and SQPCR. An increase in the SQPCR signal was seen throughout immunosuppression, with a slow decrease upon the withdrawal of steroids and a faster decrease with drug treatment. SQPCR results with lung homogenates and bronchoalveolar fluids strongly correlated with each other and with cyst counts. These results warrant investigation of SQPCR for assessing treatment results of human P. carinii pneumonia infection.