Biomedical subjects

S X Hu

Publications and source records attributed to S X Hu.

At least 55 records · Page 3Linked to original sources

PubMed1994

Levels of retinoblastoma protein expression in newly diagnosed acute myelogenous leukemia.

The relationship between the level of retinoblastoma protein (RB) expression and the survival of 113 newly diagnosed acute myelogenous leukemia (AML) patients was studied. Western blotting was used to determine the level of RB protein present in peripheral blood leukemia cells and results were confirmed in 26 patients by immunohistochemistry. The leukemic cells from 22/113 AML patients (19%) contained RB protein at levels that were equal to or less than the level of RB observed in the mononuclear cell fraction of peripheral blood from normal individuals (Low RB). Levels of RB greater than that of normal blood (Elevated RB) were seen in 91 patients (81%). The median survival of patients with low RB was significantly shorter than that seen in patients with elevated RB, 12 weeks versus 40 weeks (P = .02). Remission induction frequency was 36% in low RB patients compared with 68% in AML patients with elevated RB (P = .01). Multivariate analysis showed that low RB protein level was an independent prognostic factor predictive or poor survival after allowing for other known prognostic factors. These data suggest that a low level of the RB protein at the time of diagnosis is associated with shortened survival in AML patients because of inferior response to conventional therapy. Monitoring of the RB level could identify a subgroup of AML patients with an extremely poor prognosis when treated with chemotherapy alone, who would be eligible for alternative therapeutic strategies.

Adolescent↗

PubMed1994

Further characterization of retinoblastoma gene-mediated cell growth and tumor suppression in human cancer cells.

We have transfected the osteosarcoma cell line Saos2 and the bladder carcinoma cell line 5637 with additional retinoblastoma (RB) expression plasmids. The RB-reconstituted Saos2 and 5637 cells showed only slightly lower ratios of cells undergoing DNA synthesis compared to their parental RB- tumor cells, and there were no noticeable changes in cell morphology. Furthermore, we have isolated long-term RB+ clones from Saos2, 5637, and the retinoblastoma cell line WERI-Rb27 after transfection/transduction with a RB expression plasmid or retrovirus. These clones were similar to their parental cell lines in terms of morphology and growth rates, and they all expressed functional RB protein (p110RB) as evidenced by its potential of phosphorylation, simian virus 40 large tumor antigen binding, and nuclear tethering. No mutation or deletion of the exogenous RB gene was detectable by PCR and single-strand conformation polymorphism analysis. In addition, either the individual or pooled RB+ clones did form malignant tumors in nude mice but usually with a longer latency period and lower frequency. Such tumors also retained normal RB expression, suggesting that at least a portion of the RB-reconstituted tumor cells were still tumorigenic. This phenomenon is referred to by us as tumor suppressor resistance (TSR).

Animals↗

PubMed1994

Altered retinoblastoma protein expression and prognosis in early-stage non-small-cell lung carcinoma.

BACKGROUND: Altered retinoblastoma (RB [also known as RB1]) gene expression was initially found in a small cohort study to occur in five (22%) of 23 patients with primary stage I and II non-small-cell lung carcinomas (NSCLCs). Putative mutation of the p53 gene (also known as TP53) has also been found to occur frequently in stage I and II NSCLCs and to be associated with more aggressive disease and a poorer prognosis. PURPOSE: Our purpose was to determine the Rb protein status in the same cohort that had been previously studied for their p53 protein status and to document whether loss of Rb protein expression was also an important factor in overall survival. METHODS: One hundred one stage I or II NSCLC specimens were analyzed by immunohistochemical staining. These paraffin-embedded tumor sections were obtained from individual paraffin blocks prepared for each patient in the previous study. Patient survival status was obtained from hospital and tumor registry records. RESULTS: Altered Rb protein expression was found in 24 of 101 stage I and II NSCLCs. The median survival was 32 months for patients with Rb-positive (Rb+) tumors and 18 months for individuals in whom expression of Rb protein was absent or altered (Rb-) in tumor cells. Log-rank analysis of the differences in overall survival was statistically significant (P = .007). When these results were combined with the p53 status in the same tumor, the median survival was 12 months for those individuals who had theoretically the worst pattern (Rb-/p53+) and 46 months for those patients with theoretically the best pattern (Rb+/p53-) (P < .001). The Rb+ and Rb- groups in this cohort were well balanced with respect to the distribution of age, disease stage, histologic types, p53 status, and sex. Using a multivariate proportional hazards regression model, both altered Rb and p53 status were found to be significantly associated with poor prognosis (P = .005 and .012, respectively) in the overall cohort. CONCLUSION: Altered Rb protein expression is an independent prognostic marker for overall decreased survival in early-stage NSCLC as detected by absence of nuclear Rb protein staining. There appears to be a poorer prognosis when loss of Rb protein function and mutated p53 protein occur in the same tumor. IMPLICATIONS: If these findings can be confirmed in larger prospective studies, the results would suggest that both the Rb and p53 status should be utilized as independent prognostic factors in early-stage NSCLC.

Adenocarcinoma↗

PubMed1994

Loss of the retinoblastoma tumor-suppressor gene in parathyroid carcinoma.

BACKGROUND: The origin and molecular pathogenesis of parathyroid carcinoma are unknown. This life-threatening cause of primary hyperparathyroidism cannot be reliably distinguished from its benign counterpart on the basis of histopathological features alone. Because the PRAD1, or cyclin D1, gene, a cell-cycle regulator, has been implicated in a subgroup of benign parathyroid tumors, we examined the possibility that another cell-cycle regulator with possible functional links to PRAD1, the retinoblastoma tumor-suppressor gene (RB), might be involved in the molecular pathogenesis of parathyroid carcinoma. METHODS: Parathyroid carcinomas from 9 patients and adenomas from 21 were studied for evidence of tumor-specific loss of RB gene DNA (allelic loss) by analysis of four DNA polymorphisms and for evidence of altered expression oF RB protein by immunohistochemical staining. RESULTS: All of 11 specimens from 5 patients with parathyroid carcinoma and informative DNA patterns and 1 of 19 specimens from 19 patients with parathyroid adenoma and informative DNA patterns lacked an RB allele. Fourteen of 16 specimens (88 percent) from the nine patients with carcinoma had abnormal expression of RB protein (a complete or predominant absence of nuclear staining for the protein). None of the 19 adenomas, including the tumor with loss of an RB allele, had unequivocally abnormal staining for RB protein. CONCLUSIONS: Inactivation of the RB gene is common in parathyroid carcinoma and is likely to be an important contributor to its molecular pathogenesis. The presence of such inactivation may help to distinguish benign from malignant parathyroid disease and may have useful diagnostic, prognostic, and therapeutic implications.

Adenoma↗

PubMed1994

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.

Alleles↗

PubMed1994

Evidence of functional RB protein in epithelial ovarian carcinomas despite loss of heterozygosity at the RB locus.

The presence of retinoblastoma (RB) protein was evaluated by immunohistochemical staining and correlated with loss of heterozygosity (LOH) at the RB locus in 52 primary epithelial ovarian carcinomas. Forty-eight tumors were informative at the RB locus by molecular genetic analysis. Twenty-five tumors (52%) showed loss of heterozygosity at the RB locus. RB protein expression was found in 23 of these tumors. The remaining two tumors were negative for RB protein product by immunohistochemical staining. All 23 tumors showing no LOH at the RB locus had a normal RB protein pattern. All but three tumors revealed either no LOH with any marker or, if LOH was found for one chromosome 13 marker, all other informative markers also showed LOH. The three recombinant tumors included two which retained alleles at one or more loci distal and one which retained alleles proximal to the RB locus. LOH at the RB locus was significantly more common in invasive high-grade (grades 3 and 4) tumors as compared to invasive low-grade (grades 1 and 2) tumors (P < 0.001). Our data suggest that while molecular genetic studies reveal frequent LOH at the RB locus, particularly in high-grade tumors, normal RB protein expression is present in the majority (96%) of these tumors. This implies that another, unidentified, gene or genes located on chromosome 13 may be important in the progression of most epithelial ovarian carcinomas. Additionally, it is likely that the specific chromosome 13 alteration(s) associated with sporadic ovarian neoplasms will be extremely difficult to identify using allelic loss and deletion mapping studies.

Epithelium↗

PubMed1993

Loss of RB protein expression in primary bladder cancer correlates with loss of heterozygosity at the RB locus and tumor progression.

RB-protein status as determined by immunohistochemical analysis was compared with loss of heterozygosity (LOH) at the RB locus in 68 primary transitional-cell carcinomas of the bladder. Absence of RB-protein expression was found in 15 of 17 tumors in which LOH at the RB locus was identified, whereas 31 of 36 tumors from informative patients which showed no LOH had a normal RB-protein pattern (p < 0.001). Altered RB-protein expression was also more frequently seen in muscle-invasive and high-grade tumors (p < 0.003 and < 0.005, respectively). Our results indicate that LOH at the RB locus is highly correlated with loss of RB-protein expression in primary bladder carcinomas and further strengthens the notion that loss of RB function may be associated with more aggressive bladder tumors.

Carcinoma, Transitional Cell↗

PubMed1992

Changes in growth and tumorigenicity following reconstitution of retinoblastoma gene function in various human cancer cell types by microcell transfer of chromosome 13.

Functional loss of the retinoblastoma (RB) gene has been implicated in the initiation or progression of several human tumor types including cancer of the eye, bone, bladder, and prostate. To examine the consequence of adding one RB allele containing its normal regulatory elements back into representative examples of each of these cancer types, as well as to compare the results to those previously reported using various RB complementary DNA constructs, a neomycin resistant marked 13 chromosome was transferred by microcell fusion. Several attempts to obtain RB positive osteosarcoma cells failed. In addition, only one RB positive retinoblastoma clone was isolated. This clone contained many large cells, could not be maintained in long-term culture, and produced only RB negative tumors. Three RB positive bladder cancer cell clones were obtained, all of which grew slower in culture than their RB negative parental counterpart and did not form colonies in soft agar. Tumorigenicity was markedly suppressed in these clones. One clone yielded no tumors, and the other 2 clones produced only one small tumor each, both of which were RB negative. In contrast, the 2 RB positive prostate cancer cell clones isolated had no differences in their cell culture growth properties, including growth in soft agar compared to the parental cells. One of the clones was nontumorigenic, while the other clone produced 4 small tumors, all of which were RB positive. These results indicate that the transfer of one RB allele by microcell transfer produces different levels of growth inhibition as well as tumor suppression, depending on the cell type examined. In the case of prostate cancer, the function of the RB gene in tumor suppression appears to be independent from its growth regulatory function, since no growth inhibition in cell culture was noted in these cells, although tumor suppression was significant.

Alleles↗

PubMed1992

Clinical implications of decreased retinoblastoma protein expression in acute myelogenous leukemia.

The retinoblastoma (RB) protein levels in blast-enriched mononuclear fractions from the peripheral blood of 33 newly diagnosed patients with acute myelogenous leukemia were studied. Ten patients who had previously been treated were also analyzed, nine of whom had achieved prior complete remission. Low RB protein expression was found in 13 of 43 (30%) of the acute myelogenous leukemia patients as determined by Western blotting and immunochemical analysis. Of particular interest among the 20 newly diagnosed patients treated with the same therapeutic regimen, the median survival was 39 days for those with low RB protein expression compared to 333 days for those with high levels of RB protein expression in their leukemic cells (P less than or equal to 0.02). This preliminary study suggests that decreases of RB protein expression in peripheral blood of myeloid leukemic cells occur frequently and may be associated with shortened survival of acute myelogenous leukemia patients.

Blotting, Western↗

PubMed1992

Altered expression of retinoblastoma protein and known prognostic variables in locally advanced bladder cancer.

BACKGROUND: The clinical behavior of the tumor in patients with locally advanced bladder carcinoma is unpredictable. Current predictors of clinical behavior include depth of muscle invasion, presence of vascular invasion, proliferation rate, and loss of blood group antigens. Treatment selection would be facilitated by the development of a reliable marker of tumor progression. Functional retinoblastoma (RB) gene loss has been reported to occur in bladder carcinoma, but the significance of this loss is unknown. PURPOSE: We have evaluated the frequency of functional loss of the RB gene in locally advanced bladder carcinoma and have compared the results to known prognostic factors in the same cohort. METHODS: Forty-three study patients with pathologically well-characterized, locally advanced bladder carcinoma, who were placed in a protocol incorporating surgery and chemotherapy, were studied for known clinical and pathological prognostic indicators as well as for their Rb status. Formalin-fixed and paraffin-embedded archival primary tumor tissues were used for histological and immunohistochemical analyses. RESULTS: Altered Rb protein expression was documented in 37% of the tumor specimens. The high rate of altered Rb expression found in this cohort with advanced urothelial tumors strongly suggests that RB functional loss may be associated with tumor progression in this malignancy. Altered Rb protein expression was found to be independent of other known prognostic variables. A significantly poorer tumor-free survival rate also was noted for those patients who had a tumor with an altered Rb protein with or without vascular invasion. CONCLUSION: The high frequency of Rb alteration in locally advanced bladder carcinomas, plus the fact that a significant correlation could not be found between the Rb status and other known prognostic markers in this preliminary study, suggests that altered RB expression may be an independent prognostic marker of tumor progression in bladder cancer.

Gene Expression Regulation, Neoplastic↗

PubMed1992

Human cytomegalovirus-stimulated peripheral blood mononuclear cells induce HIV-1 replication via a tumor necrosis factor-alpha-mediated mechanism.

Human cytomegalovirus (HCMV) is a potential cofactor in HIV-1 infection. To investigate the mechanism whereby HCMV promotes HIV-1 replication, a PBMC coculture assay which measures HIV-1 p24 antigen release was used as an index of viral replication. HCMV-stimulated PBMC were capable of inducing HIV-1 replication in cocultures with acutely infected PBMC; however, this occurred only when the PBMC were from HCMV-seropositive donors (598 +/- 207 versus 27 +/- 10 pg/ml p24 antigen with PBMC from HCMV-seronegative donors on day 6 of coculture). Upon stimulation with HCMV, PBMC obtained exclusively from HCMV-seropositive donors released tumor necrosis factor (TNF)-alpha (270 +/- 79 pg/ml at 18 h of culture). Monoclonal antibodies to TNF-alpha blocked the activity of HCMV-stimulated PBMC in cocultures both with acutely HIV-1-infected PBMC and with the chronically infected promonocytic line U1. Also, treatment of HCMV-stimulated PBMC with pentoxifylline, an inhibitor of TNF-alpha mRNA, markedly reduced HIV-1 replication in cocultures both with acutely and chronically infected cells. These results indicate that TNF-alpha is a key mediator of HIV-1 replication induced by HCMV-stimulated PBMC and support the concept that this cytokine plays an important role in the pathogenesis of HIV-1 infection.

Cells, Cultured↗

PubMed1991

Inactivation of the retinoblastoma gene in human bladder and renal cell carcinomas.

The retinoblastoma (RB) gene was the first tumor suppressor gene isolated and its inactivation is associated with the pathogenesis of several types of human cancer. In this study, we investigated the involvement of the RB gene in bladder and renal cell carcinomas by determining the loss of heterozygosity (LOH) at the RB locus and by DNA, RNA, and protein analysis of the RB gene. Whenever possible, the latter included Western blotting and immunohistochemical staining of the RB protein. In bladder carcinoma, 2 of the 8 cell lines we studied had an inactivated RB gene; one cell line lacked RB expression without a gross RB deletion, whereas the other cell line expressed only the underphosphorylated form of the RB protein. None of 16 low-grade noninvasive bladder carcinomas showed an alteration in RB protein by direct Western blot analysis, whereas 2 of 14 high-grade, invasive tumors had no RB protein as measured by both Western blotting and immunohistochemical staining. This suggests that the loss of RB function may be more important in the progression of bladder cancer than in its initiation, although more extensive studies are required. LOH within the RB locus was observed in 5 of 27 informative cases of primary bladder, ureter, or renal pelvis carcinoma. However, none of the 5 cases with LOH at the RB locus had a functional loss of RB protein expression. In renal cell carcinoma, one of the 12 cell lines had a gross homozygous deletion of the RB gene, and 2 of 32 primary tumors were negative for RB protein expression. LOH at the RB locus also was found in only 2 of 30 informative cases, one of which lacked RB expression. These results are the first to demonstrate the involvement of RB inactivation in the development of advanced primary bladder carcinoma and suggest that RB loss could have a role in certain renal cell carcinomas. Our data, however, show no correlation between LOH at the RB locus in bladder cancer and actual inactivation of the RB gene at the protein level. This may suggest that there is a second tumor suppressor or recessive cancer gene on chromosome 13 in bladder cancer and/or that the mechanism of RB inactivation in bladder cancer frequently involves independent mutations of each RB allele.

Base Sequence↗

PubMed1991

Intraocular tumor formation of RB reconstituted retinoblastoma cells.

It has been reported that replacement of a functional retinoblastoma (RB) gene in RB defective WERI-27 retinoblastoma cells results in complete loss of their tumorigenic potential in nude mice following s.c. injection. We have repeated the identical studies and found that although tumors did not develop s.c., the RB reconstituted cells, either soon after RB virus infection or after long term cultivation, consistently produced tumors when injected intraocularly. These tumor cells, when reestablished in culture, were found to retain a normal RB protein as determined by direct Western blotting and immunocytochemical staining. The tumors, however, occurred with a longer average latency period and with less frequency compared to those produced by the parental RB defective cells. Our results suggest that reintroduction of the RB gene into WERI-27 cells reduces but does not completely suppress their tumorigenic potential. Since retinoblastoma is an eye tumor it also provides further documentation that the use of an orthotopic injection site can be critical when determining the tumorigenicity of a given cell type.

Animals↗

PubMed1991

The retinoblastoma gene functions as a growth and tumor suppressor in human bladder carcinoma cells.

The product of the human retinoblastoma gene (RB) is a nuclear phosphoprotein that is thought to function as a tumor suppressor. Mutations of RB frequently occur in human bladder carcinoma. To investigate the significance of the functional loss of this gene in bladder cancer, an RB expression plasmid (pBARB) under control of the human beta-actin promoter was transfected into the bladder carcinoma cell line HTB9, which lacks RB expression. Marker-selected transfectants that expressed RB protein were identified by immunoblotting and immunohistochemical staining. In selected clones, stable RB expression has persisted over 1 yr under standard culture conditions with 10% serum. However, RB expression caused major alterations of HTB9 growth properties both in vitro and in vivo. RB+ transfectants lacked the ability to form colonies in semi-solid medium, and their growth rate was significantly decreased in 3% serum. In addition, the tumorigenicity of these transfectants was markedly decreased. Tumors that formed in nude mice were much smaller and had a longer latency period but were indistinguishable microscopically from those produced by parental cells. Slower growing tumors were RB+, as measured by nuclear staining of their RB protein and by a normal RB protein pattern on immunoblots. These findings support the concept that the RB gene acts as both a growth and tumor suppressor in bladder cancer cells.

Actins↗

PubMed1991

Absence of retinoblastoma protein expression in primary non-small cell lung carcinomas.

Retinoblastoma (RB) protein expression was examined in paraffin and frozen tissue sections of 36 primary non-small cell lung carcinomas (NSCLC) using immunohistochemistry with confirmation by direct Western blotting. A normal RB protein staining pattern was present in 24 and absent in 10 NSCLC. Two additional RB positive primary tumors have major foci in which all tumor cells showed no RB protein staining. Significantly more high-stage (stages III and IV) NSCLC had altered RB protein expression than those with low-stage (stages I and II) tumors (P less than 0.05). The results suggest that absence of the RB expression may be associated with the initiation and/or progression of many NSCLC. This is the first report of successful RB staining in paraffin sections.

Blotting, Northern↗

PubMed1991

Altered cytokine release in peripheral blood mononuclear cell cultures from patients with the chronic fatigue syndrome.

Chronic fatigue syndrome (CFS) is an idiopathic illness associated with a variety of immunologic abnormalities. To investigate potential pathogenetic mechanisms, we evaluated serum levels and peripheral blood mononuclear cell (PBMC) production of selected cytokines and immunoglobulins. Serum bioactive transforming growth factor beta (TGF-beta) levels were higher (P less than 0.01) in patients with CFS (290 +/- 46 pg/mL) than in control subjects (104 +/- 18 pg/mL), but levels of other cytokines tested were not different. Lipopolysaccharide-stimulated release of interleukin 1 beta (IL-1 beta), IL-6, and tumor necrosis factor-alpha was increased (P less than 0.05) in PBMC cultures from patients with CFS versus control subjects; enhanced (P less than 0.01) IL-6 release to phytohemagglutinin was also observed. In contrast, TGF-beta release in response to lipopolysaccharide was depressed (P less than 0.01) in PBMC cultures derived from patients with CFS. No differences in IL-2 and IL-4 or immunoglobulin production were observed. The enhanced release of inflammatory cytokines by stimulated PBMC from patients with CFS suggests that these cells are primed for an increased response to immune stimuli. These data also suggest an association between abnormal regulation of TGF-beta production in vivo and in vitro with the immunologic consequence of CFS.

Cytokines↗

PubMed1991

Lack of nuclear RB protein staining in G0/middle G1 cells: correlation to changes in total RB protein level.

Functional loss of the retinoblastoma (RB) gene has been implicated in a diverse group of human malignancies. The potential clinical importance of such loss demands a reproducible RB cell staining method to distinguish RB+ from RB- tumors. We report that normal and known RB+ tumor cells either in culture or in vivo display a mosaic RB staining pattern with variable proportions of cells having unstained nuclei which are indistinguishable from truly RB- tumor cells. We also show that the apparent lack of nuclear staining corresponds to RB+ cells which are in the G0 or middle G1 phase, where as much as 10-fold decrease in total cellular RB protein occurs compared to their G2/M phase counterparts. These findings not only provide new clues to RB protein function in cell growth and differentiation, but also need to be considered when evaluating the RB status in primary malignancies for its role in tumor initiation/progression or as a prognostic parameter.

Animals↗

PubMed1991

Characterization of intragenic deletions in two sporadic germinal mutation cases of retinoblastoma resulting in abnormal gene expression.

Two gross intragenic deletions of the retinoblastoma (RB) gene from patients with sporadic hereditary disease were analysed in detail. This study was undertaken to determine whether there were common mechanisms for these deletions and whether changes found in the tumors were identical to those found in the constitutional cells. Short repeats at the deletion breakpoints were found in both tumors, one resulting in a 2.0 kb deletion including exons 21 and 22, and the other producing a 3.7 kb deletion including exons 14-17. In addition, the identical sequence changes documented in genomic DNAs and transcripts of these tumors were also observed in the constitutional cells. Both deletions were in-frame and resulted in a truncated transcript and protein identical to the expected size based on the exons deleted. These studies provide further documentation of the events which represent the actual 'two hits' originally hypothesized to be responsible for retinoblastoma development.

Base Sequence↗