Biomedical subjects
Publications and source records attributed to M B Wang.
Gene silencing was perceived initially as an unpredictable and inconvenient side effect of introducing transgenes into plants. It now seems that it is the consequence of accidentally triggering the plant's adaptive defence mechanism against viruses and transposable elements. This recently discovered mechanism, although mechanistically different, has a number of parallels with the immune system of mammals.
BACKGROUND: Parathyroid adenoma is the most common cause of primary hyperparathyroidism (pHPT). Adenomas usually involve only a single gland, and the remaining glands are normal or suppressed. Multiple parathyroid adenomas have been reported to occur in as high as 11% of patients with pHPT. The significant incidence of multiple adenomas with histologic similarities to hyperplasia has raised the possibility that adenoma is a continuation of the hyperplasia state. To test this theory, we used molecular genetics to compare clonality and proliferative activity of parathyroid adenoma with its corresponding normal glandular tissue. Furthermore, we devised a scheme to definitively distinguish between the different parathyroid states on a molecular level, because histologic distinction is unreliable. METHODS: The study included three patients with a diagnosis of singular parathyroid adenoma and three with double parathyroid adenomas. Paraffin-embedded surgical specimens of both adenomas and normal glands were retrieved from each patient. Clonal analysis of the phosphoglycerolkinase (PGK) gene has suggested that parathyroid adenomas are monoclonal. Clonality of parathyroid adenomas and normal parathyroid glands was studied by polymerase chain reaction-based restriction fragment length polymorphic analysis for the PGK gene. Proliferative activity of the specimens was also analyzed using the immunohistochemical markers PCNA and Ki-67. RESULTS: All adenomas were monoclonal and all normal parathyroid glands were polyclonal for the PGK gene in both the single and double adenoma specimens. All adenomas stained positive for proliferative activity. In the three patients with singular adenoma, proliferative activity was not detected in the normal parathyroid tissue. However, in the double adenoma group, two of the three patients showed hyperproliferative activity in the normal glands. CONCLUSION: Proliferative activity consistent with hyperplasia was present in some normal glands of multiple adenoma patients. Our observation supports the theory that multiple adenomas may be a continuation of the hyperplasia state.
Recent research has revealed the existence of an elegant defence mechanism in plants and lower eukaryotes. The mechanism, known in plants as post-transcriptional gene silencing, works through sequence-specific degradation of RNA. It appears to be directed by double-stranded RNA, associated with the production of short 21-25 nt RNAs, and spread through the plant by a diffusible signal. The short RNAs are implicated as the guides for both a nuclease complex that degrades the mRNA and a methyltransferase complex that methylates the DNA of silenced genes. It has also been suggested that these short RNAs might be the mobile silencing signal, a suggestion that has been challenged recently.
Tobacco plants were transformed with a chimeric transgene comprising sequences encoding beta-glucuronidase (GUS) and the satellite RNA (satRNA) of cereal yellow dwarf luteovirus. When transgenic plants were infected with potato leafroll luteovirus (PLRV), which replicated the transgene-derived satRNA to a high level, the satellite sequence of the GUS:Sat transgene became densely methylated. Within the satellite region, all 86 cytosines in the upper strand and 73 of the 75 cytosines in the lower strand were either partially or fully methylated. In contrast, very low levels of DNA methylation were detected in the satellite sequence of the transgene in uninfected plants and in the flanking nonsatellite sequences in both infected and uninfected plants. Substantial amounts of truncated GUS:Sat RNA accumulated in the satRNA-replicating plants, and most of the molecules terminated at nucleotides within the first 60 bp of the satellite sequence. Whereas this RNA truncation was associated with high levels of satRNA replication, it appeared to be independent of the levels of DNA methylation in the satellite sequence, suggesting that it is not caused by methylation. All the sequenced GUS:Sat DNA molecules were hypermethylated in plants with replicating satRNA despite the phloem restriction of the helper PLRV. Also, small, sense and antisense approximately 22 nt RNAs, derived from the satRNA, were associated with the replicating satellite. These results suggest that the sequence-specific DNA methylation spread into cells in which no satRNA replication occurred and that this was mediated by the spread of unamplified satRNA and/or its associated 22 nt RNA molecules.
Post-transcriptional silencing of plant genes using anti-sense or co-suppression constructs usually results in only a modest proportion of silenced individuals. Recent work has demonstrated the potential for constructs encoding self-complementary 'hairpin' RNA (hpRNA) to efficiently silence genes. In this study we examine design rules for efficient gene silencing, in terms of both the proportion of independent transgenic plants showing silencing, and the degree of silencing. Using hpRNA constructs containing sense/anti-sense arms ranging from 98 to 853 nt gave efficient silencing in a wide range of plant species, and inclusion of an intron in these constructs had a consistently enhancing effect. Intron-containing constructs (ihpRNA) generally gave 90-100% of independent transgenic plants showing silencing. The degree of silencing with these constructs was much greater than that obtained using either co-suppression or anti-sense constructs. We have made a generic vector, pHANNIBAL, that allows a simple, single PCR product from a gene of interest to be easily converted into a highly effective ihpRNA silencing construct. We have also created a high-throughput vector, pHELLSGATE, that should facilitate the cloning of gene libraries or large numbers of defined genes, such as those in EST collections, using an in vitro recombinase system. This system may facilitate the large-scale determination and discovery of plant gene functions in the same way as RNAi is being used to examine gene function in Caenorhabditis elegans.
BACKGROUND: Research projects are frequently presented at national meetings not only to make the data available, but also to further critically evaluate the project. The ultimate goal remains publication of the research. We assessed the publication rate of presentations at 1993-95 annual meetings. METHODS: All presentations at the scientific sessions were searched in the computerized database Melvyl MEDLINE and PubMed. The papers were categorized in 6 broad groups. RESULTS: The overall proportion of presentations that were published was 32%. Clinical papers in pediatrics had the highest publication rate, followed by basic science research in laryngology and plastics. CONCLUSION: The publication rate of 32% is lower than the rate at meetings in other fields, reflecting variability in selection criteria for the presentations and quality of abstracts presented. Basic science presentations did not have a greater publication rate as compared to clinical presentations.
OBJECTIVES: Cyclin D1 is a cell cycle regulatory factor that modulates a critical step in cell cycle control. Cyclin D1 is overexpressed in a significant proportion of head and neck cancers and correlates with a poor prognosis. Abrogation of cyclin D1 action through antisense cyclin D1 shows promise as an antitumor therapy, with an inhibitory effect in head and neck squamous cell carcinoma both in vitro and in vivo. The suppressive effect of antisense cyclin D1 in head and neck cancer xenografts in nude mice is incomplete, however, suggesting that combination with another antitumor agent is necessary for complete tumor eradication. Cisplatin is a widely used chemotherapeutic agent in head and neck cancer, and is particularly effective in combination with radiation therapy. In this study, we investigate whether antisense cyclin D1 enhances the sensitivity of head and neck cancer cells to cisplatin. Such an enhancement of sensitivity would suggest that combination therapy using antisense cyclin D1 and cisplatin would be an effective treatment modality for head and neck cancer. STUDY DESIGN: Antisense cyclin D1 was transfected into the head and neck squamous cell carcinoma cell line CCL23 using a plasmid vector. Both the parental CCL23 cells and the antisense cyclin D1-transfected CCL23 cells (CCL23AS) were treated with cisplatin at increasing concentrations. The dosage of cisplatin ranged from 1 microg/mL to 10 microg/mL. Initial exposure to cisplatin was for 2 hours, with increasing exposure times in succeeding experiments. Cell viability assays were done following cisplatin exposure. Dose response curves for the two cell lines were plotted and compared. Western blot analyses were done on the cisplatin-treated cell lines to determine levels of cyclin D1 expression. RESULTS: Increasing concentrations of cisplatin resulted in significantly higher rates of cell killing in the antisense cyclin D1-transfected cells than in the parental cells. The ID50 values for the parental CCL23 cells and the antisense cyclin D1-transfected CCL23 cells were 7 microg/mL and 3 microg/mL, respectively, indicating significant enhancement of sensitivity to cisplatin in the antisense cyclin D1-transfected cells. Western blot analyses demonstrated decreased expression of cyclin D1 in the CCL23AS cells with increasing doses of cisplatin, compared with the parental CCL23 cells. CONCLUSIONS: Antisense cyclin D1-transfected CCL23 cells demonstrate an enhanced sensitivity to the effects of cisplatin compared with the parental cell line. Although the mechanism for this phenomenon is not completely understood, the data suggests the potential use of combination therapy using antisense cyclin D1 and cisplatin for head and neck cancers. While neither agent alone can completely eradicate head and neck cancers, the synergistic effect of the two may be an effective therapeutic protocol for refractory head and neck cancers. Future investigation into the combination of antisense cyclin D1 with cisplatin for treatment of head and neck cancer is needed.
OBJECTIVE: To test whether nitric oxide (NO) enhances the cytotoxicity of cisplatin in a head and neck squamous cell carcinoma (HNSCC) cell line. BACKGROUND: Cisplatin is one of the most frequently used chemotherapeutic agents in the treatment of HNSCC. NO has been shown to play an important role in regulating tumor growth. Previous studies demonstrate that NO can enhance the cytotoxicity of cisplatin in Chinese hamster lung fibroblasts. In this report, we examined the in vitro interaction of NO and cisplatin in a HNSCC cell line. MATERIALS AND METHODS: CCL23 cells were pretreated with three different NO donors: PAPA/NO (t 1/2 = 15 min), DPTA/NO (t 1/2 = 3 h), and DETA/NO (t 1/2 = 20 h). The cells were rinsed and exposed for 6 hours to a culture medium containing cisplatin. Cell survival and LD50 of cisplatin were calculated with and without NO pretreatment. RESULTS: PAPA/NO and DPTA/NO did not show any cytotoxic activity and did not change the LD50 of cisplatin. DETA/NO when used alone resulted in 25.6% cell death at its peak dose (100 microM). Pretreatment with DETA/NO resulted in almost a threefold reduction of the LD50 of cisplatin (6.8 vs. 2.4 microg/mL). Pretreatment with DETA/NO sensitized the HNSCC cells to subsequent cisplatin activity (two-sided P =.00016). CONCLUSION: Pretreatment of HNSCC cells with long-acting NO donors enhances cisplatin activity. Short- and medium-acting NO donors do not exert a toxic effect and do not augment the activity of cisplatin. NO agonists should be considered in the future as a possible adjunct to cisplatin in the treatment of HNSCC. Further studies with animal models are necessary to further clarify this relationship.
No single method of reconstruction has proven ideal for all patients with defects following vertical hemilaryngectomy. In this report, we detail a new technique for hemilaryngeal reconstruction involving the use of a pedicled buccal mucosa island flap supplied by the facial artery and vein. The buccal flap was used to resurface a transversely oriented sternohyoid myofascial flap. The reconstructive outcome was analyzed in 4 animals, 3 of which survived the early postoperative period. Videoendoscopy and stroboscopy were performed to analyze the laryngeal configuration and vibration. Each subject was decannulated and had a competent airway free of aspiration. After sacrifice of the animals, whole organ axial sections were made at multiple levels. Endoscopic and histologic findings documented that this technique produced an appropriate neocord position. Laryngeal stroboscopy in each animal showed bilateral mucosal traveling waves, with entrainment of the reconstructed neocord mucosa and native vocal cord mucosa. We conclude that the layered reconstructive technique described, compared to traditional methods of reconstruction, more closely replicates the structure of the excised tissue in hemilaryngeal reconstruction, potentially resulting in an improved voice outcome.
PROBLEM: Cyclin D1 is a regulatory factor essential in the progression of the cell cycle from G1 through S phase. Amplification and overexpression of cyclin D1 have been observed in many human cancers including head and neck squamous cell carcinoma (HNSCC). We have previously transfected a HNSCC control cell line (CCL23) with an antisense cyclin D1 plasmid and demonstrated inhibition of cell proliferation in vitro. In this study, we examine whether antisense cyclin D1 could inhibit tumor growth in vivo. Methods/measures: The CCL23 and its antisense cyclin D1 transfected clone (CCL23 AS) were injected into the flanks of nude mice. Tumor growth was monitored weekly. After 5 weeks, tumors were removed and studied for tumor size, cyclin D1 expression, cyclin D1-dependent kinase activity, and retinoblastoma (Rb) phosphorylation. RESULTS: Compared with the control tumors, 11 of 19 antisense tumors were smaller, 7 tumors were of equal size, and 1 tumor was larger. Immunohistochemical analysis with an anti-cyclin D1 antibody demonstrated decreased cyclin D1 expression in CCL23 AS and the smaller antisense tumors. Cyclin D1-dependent kinase activity was reduced in CCL23 AS and the smaller antisense tumors, and this was accompanied by a relative decrease in phosphorylated Rb in these samples. CONCLUSION: Antisense cyclin D1 inhibits growth of HNSCC tumors. Cyclin D1 expression, cyclin D1-dependent kinase activity, and Rb phosphorylation are decreased in these tumors. CLINICAL SIGNIFICANCE: These findings lend support for the potential use of antisense cyclin D1 as gene therapy for HNSCC.
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BACKGROUND: MRI-guided procedures have previously been limited by technical difficulties, including the need for MRI-compatible instruments, slow image acquisition time, and the closed nature of conventional MRI scanners. The development of open configuration MRI systems with in-room, contemporaneous imaging has greatly increased the potential for MRI-guided interventional procedures. We evaluate our clinical experience applying this technology to the head and neck. METHODS: An open design 0.2T magnet combined with an in-room monitor was used for 24 MRI-guided needle localization procedures in the head and neck. Success of the procedures was based on the ability to accurately position the instrument in the target region to allow biopsy or treatment. RESULTS: In all 24 cases placement of the instrument within the target tissue was successful. CONCLUSION: MRI-guided needle-localization procedures in an open design magnet with in-room, contemporaneous image monitoring offer advantages over previous conventional interventional MRI systems by allowing interactive guidance with near real-time imaging feedback. As a result, procedure time is reduced and accuracy of instrument positioning is increased.
Two transgenic callus lines of rice, stably expressing a beta-glucuronidase (GUS) gene, were supertransformed with a set of constructs designed to silence the resident GUS gene. An inverted-repeat (i/r) GUS construct, designed to produce mRNA with self-complementarity, was much more effective than simple sense and antisense constructs at inducing silencing. Supertransforming rice calluses with a direct-repeat (d/r) construct, although not as effective as those with the i/r construct, was also substantially more effective in silencing the resident GUS gene than the simple sense and antisense constructs. DNA hybridisation analyses revealed that every callus line supertransformed with either simple sense or antisense constructs, and subsequently showing GUS silencing, had the silence-inducing transgenes integrated into the plant genome in inverted-repeat configurations. The silenced lines containing i/r and d/r constructs did not necessarily have inverted-repeat T-DNA insertions. There was significant methylation of the GUS sequences in most of the silenced lines but not in the unsilenced lines. However, demethylation treatment of silenced lines with 5-azacytidine did not reverse the post-transcriptional gene silencing (PTGS) of GUS. Whereas the levels of RNA specific to the resident GUS gene were uniformly low in the silenced lines, RNA specific to the inducer transgenes accumulated to a substantial level, and the majority of the i/r RNA was unpolyadenylated. Altogether, these results suggest that both sense- and antisense-mediated gene suppression share a similar molecular basis, that unpolyadenylated RNA plays an important role in PTGS, and that methylation is not essential for PTGS.
OBJECTIVE: The goal was to review our experience in the management of carotid body tumors at a tertiary referral center. METHODS: A retrospective review was performed of patients at University of California-Los Angeles Medical Center in whom carotid body tumor was diagnosed between 1973 and 1998. RESULTS: Twenty-nine patients with 36 carotid body tumors were identified. Thirty-five operations were performed. Seventeen patients underwent preoperative embolization. The blood loss for these patients was less than for those without embolization. Five patients had preoperative cranial nerve deficits. Neurologic deficits were noted in 41% of patients immediately after surgery. In 24% of patients, the deficits were permanent. CONCLUSION: Surgical resection is the treatment of choice for carotid body tumors. Embolization immediately before surgery decreases blood loss and facilitates tumor removal. In our series, the risk of new postsurgical cranial nerve deficits was small. Observation of these tumors is not recommended because progressive growth is associated with increased risk of neurologic deficits.
OBJECTIVES: Fetal wound healing occurs without scar tissue formation. Differences in growth factor expression between fetal and adult fibroblasts have been explored. Recently we used RNA expression studies to demonstrate that transforming growth factor (TGF)-beta1, acidic fibroblast growth factor (alpha-FGF), and basic fibroblast growth factor (beta-FGF) could be detected in both fetal and adult fibroblast cell lines. In addition, adult fibroblasts showed greater relative expression of these growth factors than fetal fibroblasts. The aim of this study was to identify the level of protein expression in fetal and adult fibroblasts. STUDY DESIGN/METHODS: Fetal and adult fibroblasts were grown in culture using standard and serum-free media. After protein extraction, Western blot studies were performed to detect the presence and amount of TGFbeta-1, alpha-FGF, and beta-FGF growth factor proteins. beta-Tubulin was used as a control. RESULTS: TGFbeta-1, alpha-FGF, and beta-FGF proteins were detected in fetal and adult fibroblasts grown in standard and serum-free media. The fetal fibroblasts showed higher levels of all three growth factor proteins compared with the adult fibroblasts. CONCLUSIONS: Western blot studies suggest higher levels of TGFbeta-1, alpha-FGF, and beta-FGF expression in fetal fibroblasts. It is clear that significant differences exist in the expression/production of these growth factors and it seems likely that further study of these differences will help elucidate the unique healing capabilities of fetal fibroblasts.
BACKGROUND: We have previously described our treatment algorithm for patients with small head and neck cancers with advanced cervical metastases (stage N2 or greater). Primary radiotherapy is given to the primary site and neck, followed 6 weeks later with endoscopy and biopsy of the primary site. If biopsy of the primary site is negative by frozen section, an immediate neck dissection is performed even when no clinical residual neck disease is present. Our initial review found that 36% of patients with a complete clinical response to radiotherapy had positive nodes on histological examination. STUDY DESIGN: Retrospective. METHODS: The medical records of 71 patients treated at UCLA Medical Center from 1986 to 1999 by this algorithm were reviewed. RESULTS: After radiotherapy, 69 of 71 patients had a complete response at their primary site. Forty-two patients had a complete clinical response in the neck. Seventy-one neck dissections were performed. Overall, 31 of 71 neck dissections (44%) had positive nodes. Among the 42 patients with a complete response to radiotherapy, 13 (31%) had positive histological nodes. Among the 29 patients with a partial response to radiotherapy, 17 (59%) had positive nodes. Follow-up and incidence of neck recurrence are discussed. CONCLUSION: Planned neck dissection for advanced cervical metastases remains controversial for patients with a complete clinical response to radiotherapy. However, our results suggest that clinical assessment after radiotherapy cannot assure the absence of neck disease. Until there are reliable methods to distinguish which patients are truly free of neck disease, we believe the benefits of a planned neck dissection outweigh the low morbidity of this procedure.
BACKGROUND: Fetal wounds can heal without any histological evidence of scarring. Fetal wounds lack the inflammatory infiltrate characteristic of adult wounds, and the fetal environment is not necessary for scarless healing to occur. Recent evidence suggests that fibroblasts are the main effector of scarless healing in fetal tissue. What has not been shown is what profile of growth factors the fibroblast uses to influence wound repair. OBJECTIVE: To determine the expression of growth factors (transforming growth factors beta1, beta2, and beta3; acidic and basic fibroblast growth factors; keratinocyte growth factor; and platelet-derived growth factor AA, BB, and AB) of fetal and adult fibroblasts in vitro. DESIGN: Adult and fetal fibroblasts were grown in culture, and messenger RNA was extracted by standard techniques. Northern hybridization was used to identify messenger RNA transcripts for the aforementioned growth factors. Densitometry was used to compare growth factor messenger RNA expression with that of a ubiquitously expressed control, glyceraldehyde phosphate dehydrogenase. RESULTS: The data suggest that fetal and adult fibroblasts express acidic and basic fibroblast growth factor and transforming growth factor beta1. Adult fibroblasts show twice the relative expression of these growth factors compared with fetal fibroblasts. CONCLUSIONS: The adult fibroblasts demonstrate a relative excess production of cytokines compared with fetal fibroblasts. This is thought to contribute to suboptimal wound healing in adult wounds compared with the scarless healing of fetal wounds.
OBJECTIVE: Squamous cell carcinoma of the head and neck (HNSCC) still has one of the lowest 5-year survival rates. Despite advances in diagnosis, treatment, and research, survival rates have not improved in recent years. This report examines the utility of fluorescence in situ hybridization (FISH) in detecting chromosome 11q13 amplification in HNSCC and in evaluating the correlation between 11q13 amplification and tumor behavior. STUDY DESIGN: This study used FISH to determine the incidence of 11q13 amplification in 20 HNSCCs and 10 normal controls from the same patients. Tumor touch preparations and paraffin-embedded tissues from the same patient samples were used for comparative analysis. Both single and dual color FISH was performed. METHODS: Repetitive chromosome 11 specific alpha satellite DNA probe and chromosome 11q13 specific probe cyclin D1 were used for the FISH analysis. RESULTS: Experiments revealed amplification of chromosome 11q13 in three fresh touch preparations. FISH on paraffin tissues showed amplification in two additional samples. Intensity of amplification, as high as 20 copies per nucleus, was observed in paraffin preparations, whereas a maximum of only six copies was seen in fresh preparations. Amplification was not detected in any of the normal samples. All five cases with 11q13 amplification had metastases and four of these were from poorly differentiated tumors. In the nonamplified cases, 5 of 15 had metastases and 2 of 15 was poorly differentiated. CONCLUSIONS: The present study indicates that FISH is a useful technique for detecting molecular changes such as amplification of chromosome 11q13 in HNSCC. FISH in paraffin preparations allows for accurate measurement of intensity of amplification and makes it possible for the evaluation of a large collection of archival material. The data also suggest that 11q13 amplification is correlated with poorly differentiated tumors and metastasis. Thus FISH has the potential to be a valuable diagnostic/prognostic tool in head and neck cancers.