Target gene specificity of E2F and pocket protein family members in living cells.

E2F-mediated transcription is thought to involve binding of an E2F-pocket protein complex to promoters in the G(0) phase of the cell cycle and release of the pocket protein in late G(1), followed by release of E2F in S phase. We have tested this model by monitoring protein-DNA interactions in living cells using a formaldehyde cross-linking and immunoprecipitation assay. We find that E2F target genes are bound by distinct E2F-pocket protein complexes which change as cells progress through the cell cycle. We also find that certain E2F target gene promoters are bound by pocket proteins when such promoters are transcriptionally active. Our data indicate that the current model applies only to certain E2F target genes and suggest that Rb family members may regulate transcription in both G(0) and S phases. Finally, we find that a given promoter can be bound by one of several different E2F-pocket protein complexes at a given time in the cell cycle, suggesting that cell cycle-regulated transcription is a stochastic, not a predetermined, process.

Use of chromatin immunoprecipitation to clone novel E2F target promoters.

We have taken a new approach to the identification of E2F-regulated promoters. After modification of a chromatin immunoprecipitation assay, we cloned nine chromatin fragments which represent both strong and weak in vivo E2F binding sites. Further characterization of three of the cloned fragments revealed that they are bound in vivo not only by E2Fs but also by members of the retinoblastoma tumor suppressor protein family and by RNA polymerase II, suggesting that these fragments represent promoters regulated by E2F transcription complexes. In fact, database analysis indicates that all three fragments correspond to genomic DNA located just upstream of start sites for previously identified mRNAs. One clone, ChET 4, corresponds to the promoter region for beclin 1, a candidate tumor suppressor protein. We demonstrate that another of the clones, ChET 8, is strongly bound by E2F family members in vivo but does not contain a consensus E2F binding site. However, this fragment functions as a promoter whose activity can be repressed by E2F1. Finally, we demonstrate that the ChET 9 promoter contains a consensus E2F binding site, can be activated by E2F1, and drives expression of an mRNA that is upregulated in colon and liver tumors. Interestingly, the characterized ChET promoters do not display regulation patterns typical of known E2F target genes in a U937 cell differentiation system. In summary, we have provided evidence that chromatin immunoprecipitation can be used to identify E2F-regulated promoters which contain both consensus and nonconsensus binding sites and have shown that not all E2F-regulated promoters show identical expression profiles.

Computer-assisted identification of cell cycle-related genes: new targets for E2F transcription factors.

The processes that take place during development and differentiation are directed through coordinated regulation of expression of a large number of genes. One such gene regulatory network provides cell cycle control in eukaryotic organisms. In this work, we have studied the structural features of the 5' regulatory regions of cell cycle-related genes. We developed a new method for identifying composite substructures (modules) in regulatory regions of genes consisting of a binding site for a key transcription factor and additional contextual motifs: potential targets for other transcription factors that may synergistically regulate gene transcription. Applying this method to cell cycle-related promoters, we created a program for context-specific identification of binding sites for transcription factors of the E2F family which are key regulators of the cell cycle. We found that E2F composite modules are found at a high frequency and in close proximity to the start of transcription in cell cycle-related promoters in comparison with other promoters. Using this information, we then searched for E2F sites in genomic sequences with the goal of identifying new genes which play important roles in controlling cell proliferation, differentiation and apoptosis. Using a chromatin immunoprecipitation assay, we then experimentally verified the binding of E2F in vivo to the promoters predicted by the computer-assisted methods. Our identification of new E2F target genes provides new insight into gene regulatory networks and provides a framework for continued analysis of the role of contextual promoter features in transcriptional regulation. The tools described are available at http://compel.bionet.nsc.ru/FunSite/SiteScan.html.

Expression patterns of the E2F family of transcription factors during mouse nervous system development.

The E2F family of transcription factors consists of two subgroups termed E2F and DP. E2F is required for cell proliferation, and is necessary for fruit fly development. E2F activity is a target for regulation by the retinoblastoma gene family, which includes pRB, p107 and p130. Mutant RB-/-, RB-/-:p107-/- and p107-/-:p130-/- mice develop abnormally, probably as a result of dysregulation in the activity of E2F, indicating the importance of E2F in mammalian development. To investigate the role of E2F in murine development, we have examined the patterns of expression of E2F-1 through E2F-5, and DP-1 in the developing nervous system by in situ hybridization. E2F-1, E2F-2 and E2F-5 are first detected in the 9.5 days post-coitus (dpc) forebrain. Expression of these E2F forms extends caudally thereafter and includes the developing brain and the upper half of the 10.5 dpc spinal cord. By 11.5 dpc, these E2F factors are expressed throughout the central nervous system. In 12.5 dpc embryos, E2F-1, E2F-2 and E2F-5 are highly expressed in proliferating, undifferentiated neuronal precursors. As neurons differentiate and migrate to the outer marginal zones in the nervous system, expression of these E2F members is extinguished. In the developing retina, another neuronal tissue, E2F-1 expression is also confined to the proliferating, undifferentiated retinoblastic layer. In contrast, E2F-3 expression is up-regulated as retinoblasts differentiate into the ganglion cell layer. In non-neuronal tissues, high E2F-4 transcript levels are present in regions corresponding to proliferative chondrocytes, whereas E2F-2 and E2F-4 transcripts are very abundant in the thymic cortex, which contains immature thymocytes. We conclude that individual E2F forms are differentially regulated during the development of distinct tissues, and especially during neuronal development.

Characterization of the 3' untranslated region of mouse E2F1 mRNA.

The E2F family of transcription factors has been implicated in the regulation of the G1 to S phase transition of the mammalian cell cycle. We have focused on characterizing the cell cycle stage-specific expression of one family member, E2F1. Previous studies indicated that there are two mouse E2F1 (mE2F1) mRNA species whose abundance peaks in early S phase. However, it was unknown as to what constituted the structural difference between the two mE2F1 mRNAs and whether or not they encoded identical proteins. We have now cloned sequences corresponding to the 3' untranslated region (3'-UTR) of the mE2F1 gene. Northern blot analyses using different probes demonstrated that the two E2F1 mRNAs were distinguished by differences in the length of their 3' UTRs. We found that the longer (2.7-kb) mE2F1 mRNA contained two consensus RNA instability elements that the shorter (2.2-kb) mE2F1 mRNA lacked. However, a comparison of the stability of the 2.7-kb and the 2.2-kb mE2F1 mRNAs suggests that both mE2F1 mRNAs are fairly stable, having a half-life of 6-9h in both asynchronously growing cells and in the S phase of synchronized cells. Thus, we have determined that both mE2F1 mRNAs contain the identical coding region of the E2F1 protein and that enforced expression of mE2F1 mRNA should not be hampered by problems with RNA stability.

Acute and chronic morbidity differences between muscle-sparing and standard lateral thoracotomies.

INTRODUCTION: Opinions differ regarding differences between totally muscle-sparing thoracotomy and standard lateral thoracotomy approaches to pulmonary resection with respect to operative time, postoperative pain and morbidity, and occurrence of chronic postthoracotomy pain syndromes and subjective shoulder dysfunction. METHODS: Three hundred thirty-five consecutive patients undergoing muscle-sparing thoracotomy (n = 148) or lateral thoracotomy (n = 187) to accomplish lobectomy for stage I lung cancer during a 40-month period were evaluated. Local rib resection was not employed, and two chest tubes were routinely used after operation in both thoracotomy groups. Epidural analgesia use was similar after operation in the two groups (muscle-sparing thoracotomy 38%, lateral thoracotomy 38%). The postoperative hospital courses and patient functional statuses at 1 year were examined. RESULTS: Demographic analyses demonstrated no differences between groups in age, sex, or association of significant comorbid medical illness. Although the operative time required for muscle-sparing thoracotomy was shorter, there were no differences between thoracotomy approaches in any of the other primary acute postoperative variables analyzed (chest tube duration, length of hospital stay, postoperative narcotic requirements, and postoperative mortality). The frequencies of chronic pain and shoulder dysfunction assessed 1 year after operation were also similar between thoracotomy groups. CONCLUSIONS: The relative efficacies and rates of occurrence of acute or chronic morbidity are equivalent after muscle-sparing thoracotomy and standard lateral thoracotomy. Although muscle-sparing thoracotomy may possibly be performed more expediently, it appears that the singular advantage of muscle-sparing thoracotomy over standard lateral thoracotomy involves the preservation of chest wall musculature in case rotational muscle flaps should be needed later.

Wedge resection versus lobectomy for stage I (T1 N0 M0) non-small-cell lung cancer.

BACKGROUND: The role of nonanatomic wedge resection in the management of stage I (T1 N0 M0) non-small-cell lung cancer continues to be debated against the present gold standard of care--anatomic lobectomy. METHODS: We analyzed the results of 219 consecutive patients with pathologic stage I (T1 N0 M0) non-small-cell lung cancer who underwent open wedge resection (n = 42), video-assisted wedge resection (n = 60), and lobectomy (n = 117) to assess morbidity, recurrence, and survival differences between these approaches. RESULTS: There were no differences among the three groups with regard to histologic tumor type. Analysis demonstrated the wedge resection groups to be significantly older and to have reduced pulmonary function despite a higher incidence of treatment for chronic obstructive pulmonary disease when compared with patients having lobectomy. The mean hospital stay was significantly less in the wedge resection groups. There were no operative deaths among patients having wedge resection; however, a 3% operative mortality occurred among patients having lobectomy (p = 0.20). Kaplan-Meier survival curves were nearly identical at 1 year (open wedge resection, 94%; video-assisted wedge resection, 95%; lobectomy, 91%). At 5 years survival was 58% for patients having open wedge resection, 65% for those having video-assisted wedge resection, and 70% for those having lobectomy. Log rank testing demonstrated significant differences between the survival curves during the 5-year period of study (p = 0.02). This difference was a result of a significantly greater non-cancer-related death rate by 5 years among patients having wedge resection (38% vs 18% for those having lobectomy; p = 0.014). CONCLUSION: Wedge resection, done by open thoracotomy or video-assisted techniques, appears to be a viable "compromise" surgical treatment of stage I (T1 N0 M0) non-small-cell lung cancer for patients with cardiopulmonary physiologic impairment. Because of the increased risk for local recurrence, anatomic lobectomy remains the surgical treatment of choice for patients with stage I non-small-cell lung cancer who have adequate physiologic reserve.

Diagnosing the indeterminate pulmonary nodule: percutaneous biopsy versus thoracoscopy.

BACKGROUND: The malignant potential of indeterminate solitary pulmonary nodules (SPN) mandates accurate diagnostic management. METHODS: 613 patients undergoing either computed tomographic lung biopsy (CT-Bx) (n = 312) or thoracoscopic excisional biopsy (Thor-Bx) (n = 301) for the diagnosis of SPN were evaluated for relative accuracy, complications, and effect on clinical treatment. RESULTS: CT-Bx identified a malignant diagnosis (Dx) in 201 (64%) of 312 patients; 85 (42%) underwent operations. A total of 116 patients (58%) with synchronous cancer (n = 16), impaired physiologic condition, or unresectable lesions (n = 100) were not operated. Surgical treatment was deferred for 20 patients (6%) with a "specific benign" Dx and 44 physiologically impaired patients with "nonspecific benign" CT-Bx. Forty-seven patients with "nonspecific benign" Dx underwent operation. Thirty-two (68%) lesions were malignant (4 metastatic, 28 primary cancer). CT-Bx accuracy was 86% for malignant and 79 (71%) of 111) for benign lesions. Surgery was still required for 132 (82%) of 163 patients with resectable lesions. Complications occurred in 24% of patients. A specific benign or malignant Dx was obtained in 292 (96%) of 301 patients undergoing Thor-Bx. Conversion to thoracotomy for lobectomy occurred in 38 (21%) of 179 patients with lung cancer. One hundred forty-one patients with lung cancer and impaired physiologic condition and all patients with metastatic (n = 44) and benign lesions (n = 78) had thoracoscopic resection alone. Complications occurred in 22% of patients. CONCLUSIONS: Limited accuracy for benign Dx with CT-Bx requires surgical biopsy for patients with SPN with adequate physiologic reserve. Thor-Bx is a safe and accurate minimally invasive surgical approach to resectable peripheral SPN.

Diagnostic and therapeutic video-assisted thoracic surgery resection of pulmonary metastases.

BACKGROUND: Appropriateness of video-assisted thoracic surgery (VATS) pulmonary metastasectomy for curative intent has been a controversial topic. We reviewed our experience with VATS wedge resection for peripheral lung metastases to determine the efficacy and potential adverse consequences of this approach for pulmonary metastasectomy. METHODS: One hundred seventy-seven patients underwent VATS resection of pulmonary metastases. Diagnostic resection (VATS-dx) was performed for 78 patients when percutaneous biopsy was unsuccessful or not feasible. Potentially curative resections (VATS-rx) were performed for 99 patients. The histologic findings in this group included colorectal (68), renal (7), sarcoma (6), breast (4), melanoma (3), head/neck (3), lymphoma (2), uterine (1), and "other" (5). The average number of lesions resected was 1.4 (range, 1-7). RESULTS: VATS resection was successfully performed for all VATS-dx and VATS-rx patients. There were no perioperative deaths. Longitudinal follow-up demonstrated a mean survival of 18 months in the VATS-dx group and 28 months in the VATS-rx group. In the VATS-rx group, 37 (37%) of 99 were free of disease, at a mean follow-up interval of 37 months. Of the 57 recurrences, 5% were local, 26% were regional, and 69% were distant. CONCLUSIONS: Results with VATS resection of peripheral pulmonary metastases for diagnostic and potentially curative intentions appear comparable with historical results by "open" thoracotomy. Careful patient selection based on high-resolution helical CT scanning is important to avoid compromise of therapeutic intent. Conversion to thoracotomy is indicated when lesions identified preoperatively are not found or when technical problems encountered may compromise surgical margins when resecting lung metastases for potential cure.

Experience and technique of stapled mechanical cervical esophagogastric anastomosis.

BACKGROUND: Anastomotic leak from cervical esophagogastric anastomoses is a serious problem after esophagectomy. We explored the efficacy of partial or total mechanical anastomoses accomplished with the endoscopic linear cutting and stapling device as an alternative to hand-sewn anastomotic techniques. METHODS: During a 42-month period, 93 patients undergoing either transhiatal esophagectomy or a three-incisional approach to esophagectomy underwent either hand-sewn (n = 43), partial mechanical (n = 16), or totally mechanical (n = 34) cervical esophagogastric anastomoses. The occurrence of postoperative anastomotic leak and the development of postoperative anastomotic stricturing requiring dilation therapy were analyzed between these groups using chi2. RESULTS: All patients survived esophagectomy and were available for postoperative follow-up. Anastomotic leak developed in 10 patients (23%) with hand-sewn, 1 patient (6%) with partial mechanical, and 1 patient (3%) with total mechanical anastomoses (p < 0.05). Anastomotic stricture development paralleled the occurrence of anastomotic leak rate with 25 patients (58%) with hand-sewn, 3 patients (19%) with partial mechanical, and 6 patients (18%) with total mechanical anastomoses experiencing strictures requiring dilation therapy (p < 0.05). CONCLUSIONS: These results suggest that partial or mechanical cervical esophagogastric anastomoses created with the endoscopic stapling device may be superior to hand-sewn anastomotic techniques.

Bilateral anterior minithoracotomy with video assistance for lung volume reduction surgery and pulmonary metastasectomy.

Incisional access to pulmonary pathology involving both lungs has often involved bilateral standard thoracotomies, median sternotomy, and, recently, sequential lateral video-assisted thoracic surgical approaches. Significant problems are inherent to each of these approaches. We introduce a hybrid technique of bilateral simultaneous minithoracotomy with video assistance as an alternative to these other surgical approaches.

Effect of dietary intake before F-18 FDG positron emission tomographic scanning on the evaluation of a solitary pulmonary nodule.

The uptake of fluorine-18 fluorodeoxyglucose (F-18 FDG) by a malignant tumor depends on the blood glucose level. The authors present a striking case that illustrates the importance of blood glucose measurement in F-18 FDG positron emission tomographic (PET) imaging in a patient with a solitary pulmonary nodule. With the emergence of freestanding imaging centers, this case emphasizes the importance of using an objective method, such as a glucometer, to measure blood glucose levels before F-18 FDG PET imaging. Results of the initial scan were equivocal (the patient had eaten before the scan), whereas a hypermetabolic focus was clearly identified on a second scan obtained 2 days later.

Psychology and a helpful form of medical practice.

The present article is provided to acquaint psychologists with the view of osteopathic medicine as proposed in the Educational Goals of Osteopathic Medicine published by Irvin M. Korr and his group at the Texas College of Osteopathic Medicine at Fort Worth, Texas. The present article is meant to suggest a connection between the over-all objectives of the advanced form of medicine and the practice of clinical psychology by listing them. Aims are in essence similar.

A note on illusions.

The crucial factor in illusion is not in perception but rather in the assumption that all two-dimensional drawings represent two-dimensional reality. This leads in some cases to making unwarranted comparisons between parts of the figures.