Chadrick E. Denlinger

Suberoylanilide Hydroxamic Acid Induces Akt-mediated Phosphorylation of p300, Which Promotes Acetylation and Transcriptional Activation of RelA/p65

We have previously demonstrated that the transcription factor NF-κB is activated by histone deacetylase inhibitors in a PI3K/Akt-dependent manner. The molecular mechanisms governing this process have not been well described. By virtue of their inhibitory action, it is unclear whether the addition of histone deacetylase inhibitors simply preserves the acetylation status of RelA/p65 or whether they actively stimulate signaling cascades that result in increased acetylation and transcription of NF-κB. Here we provide evidence that suberoylanilide hydroxamic acid stimulates NF-κB transcription through a signaling cascade that involves activation of both the serine/threonine kinase Akt and the p300 acetyltransferase. Using newly developed phosphospecific antibodies to p300 (pSer1834), and site-directed mutant proteins, we find that suberoylanilide hydroxamic acid stimulates Akt activity, which is required to phosphorylate p300 at Ser1834. Akt-mediated phosphorylation of p300 dramatically increases its acetyltransferase activity as measured by an increased acetylation of RelA/p65 at Lys310, a modification that is required for full NF-κB transcription. Importantly, coordinate activation of Akt/p300 pathway by suberoylanilide hydroxamic acid occurs at the chromatin level, resulting in recruitment of activated Akt (pSer473), p300 (pSer1834), acetylated RelA/p65 (Lys310), and RNA polymerase II to the NF-κB-dependent cIAP-2 and Bfl-1/A1 promoters. These studies provide evidence that histone deacetylase inhibitors, such as suberoylanilide hydroxamic acid, not only inhibit deacetylase activity but also stimulate active NF-κB transcription and cell survival through signaling pathways involving Akt and increased p300 acetyltransferase activity.

Combined proteasome and histone deacetylase inhibition in non-small cell lung cancer

OBJECTIVE Inhibitors of histone deacetylases are potent inducers of cell-cycle arrest and apoptosis in certain malignancies. We have previously demonstrated that chemotherapy activates the antiapoptotic transcription factor nuclear factor kappa B in non-small cell lung cancer and fails to induce significant levels of apoptosis. We hypothesize that nuclear factor kappa B inhibition with the proteasome inhibitor bortezomib (formerly known as PS-341) will sensitize non-small cell lung cancer cells to histone deacetylase inhibitor-mediated apoptosis. METHODS Tumorigenic non-small cell lung cancer cells (A549, H358, and H460) were treated with bortezomib, followed by the histone deactylase inhibitor sodium butyrate. After treatment, nuclear factor kappa B transcriptional activity was measured by using a luciferase reporter assay and transcription of the nuclear factor kappa B-dependent gene IL8. Apoptosis was determined on the basis of caspase-3 activation and DNA fragmentation. Western blot analyses for the cell-cycle regulatory proteins p21 and p53 were performed, and cell-cycle alterations were determined by means of FACS analysis. Experiments were performed in triplicate, and statistical significance was determined by using unpaired t tests. RESULTS Butyrate increased nuclear factor kappa B transcriptional activity 4-fold relative to that seen in control cells (P =.05) in all non-small cell lung cancer cell lines. Treatment with bortezomib reduced butyrate-induced activation of nuclear factor kappa B to baseline levels. The proteins p21 and p53 were stabilized after treatment with bortezomib, correlating with a G(2)/M cell-cycle arrest. Treatment with butyrate alone resulted in minimal apoptosis, but combined histone deacetylase and proteasome inhibition increased apoptosis 3- to 4-fold (P =.02). CONCLUSIONS Combined molecular targeting of histone deacteylases and proteasomes synergistically induced apoptosis in non-small cell lung cancer. Pharmacologic nuclear factor kappa B suppression through proteasome inhibition, followed by treatment with histone deacetylase inhibitors, might represent a novel treatment strategy for patients with non-small cell lung cancer.

Pulmonary segmentectomy: results and complications

BACKGROUND Segmentectomy is an anatomic pulmonary parenchymal-sparing resection performed for certain benign lesions and on selected patients with lung cancer. We reviewed our experience with segmentectomy in this highly select group of patients. METHODS We retrospectively reviewed the records of 61 patients (5% of all anatomic resections) undergoing 62 segmentectomies from 1991 to 2001. Wedge resections were excluded. The operative indications were suspected or known lung cancer (43), benign disease (12), and metastatic cancer to the lung (7). Median follow-up for patients with malignancy was 28 months (range 1 to 89 months). Actuarial survival was calculated using the Kaplan-Meier method. RESULTS Segmentectomy was performed in 43 lung cancer patients with pathologic stages of Ia-22, Ib-14, IIa-2, IIb-1, IIIa/IIIb-2, and IV-2. All resection margins were negative for malignancy. Segmentectomy for benign diseases included granulomatous disease (5), pulmonary abscess (2), plasmacytoma (1), and others (4). Complications occurred in 39% (24/62) of patients, including atelectasis requiring bronchoscopy (10/62, 16%), pneumonia (9/62, 14%), air leak more than 7 days (5/62, 8%), and supraventricular dysrhythmias (6/62, 10%). In-hospital mortality was 3% (2 patients). Median length of hospital stay was 6 days (range 4 to 66 days). In the lung cancer patients the locoregional recurrence rate was 0% and the distant recurrence rate was 20%. The 4-year actuarial survival for patients with lung cancer was 72%. CONCLUSIONS Pulmonary segmentectomy has acceptable morbidity and mortality in selected patients with both benign and malignant lung disease and remains a useful procedure in a thoracic surgeons armamentarium. Distant, not locoregional recurrence, was responsible for the cancer deaths.

Acquired systemic-to-pulmonary arteriovenous malformation secondary to Mycobacterium Tuberculosis empyema

Pulmonary arteriovenous malformations (AVMs) with systemic arterial collateralization related to a prior tuberculosis empyema are extremely rare. We report the case of a 15-year-old boy who developed a pulmonary AVM with massive systemic arterial collateralization 5 years after being treated for a Mycobacterium tuberculosis empyema necessitans. The AVM was successfully managed with combined intraarterial embolization and surgical resection.

The use of gamma-irradiation and ultraviolet-irradiation in the preparation of human melanoma cells for use in autologous whole-cell vaccines

BackgroundHuman cancer vaccines incorporating autologous tumor cells carry a risk of implantation and subsequent metastasis of viable tumor cells into the patient who is being treated. Despite the fact that the melanoma cell preparations used in a recent vaccine trial (Mel37) were gamma-irradiated (200 Gy), approximately 25% of the preparations failed quality control release criteria which required that the irradiated cells incorporate 3H-thymidine at no more than 5% the level seen in the non-irradiated cells. We have, therefore, investigated ultraviolet (UV)-irradiation as a possible adjunct to, or replacement for gamma-irradiation.MethodsMelanoma cells were gamma- and/or UV-irradiated. 3H-thymidine uptake was used to assess proliferation of the treated and untreated cells. Caspase-3 activity and DNA fragmentation were measured as indicators of apoptosis. Immunohistochemistry and Western blot analysis was used to assess antigen expression.ResultsUV-irradiation, either alone or in combination with gamma-irradiation, proved to be extremely effective in controlling the proliferation of melanoma cells. In contrast to gamma-irradiation, UV-irradiation was also capable of inducing significant levels of apoptosis. UV-irradiation, but not gamma-irradiation, was associated with the loss of tyrosinase expression. Neither form of radiation affected the expression of gp100, MART-1/MelanA, or S100.ConclusionThese results indicate that UV-irradiation may increase the safety of autologous melanoma vaccines, although it may do so at the expense of altering the antigenic profile of the irradiated tumor cells.

Extensive neurocristic hamartoma with skeletal muscle involvement

Neurocristic hamartomas (NCH) are rare pigmented skin lesions based in the deep subcutaneous tissues that may be either congenital or acquired. The clinical importance of these lesions is the potential for misdiagnosis and the development of malignant melanomas over a poorly described time course. Histological pleomorphism precludes meaningful random biopsies as a means of cancer surveillance. We present the case of an extensive NCH in a 67‐year‐old man, with a reported duration of greater than 50 years and no current clinical or histological indication of malignancy. Incisional biopsies of nodular areas showed bland‐appearing pigmented cells that extended into subcutaneous adipose tissue and skeletal muscle. The specimens contained numerous clusters of differing configurations and cell types. Positron emission tomography (PET) scanning was used as an adjunct to physical examination in follow up. A PET‐avid mass was detected but proved to be a banal nodular melanocytic proliferation within the NCH. In conclusion, NCH may be characterized by extensive deep tissue involvement in the absence of overt malignancy. The possible development of malignant melanoma in such lesions warrants close surveillance.

Laparoscopic gastric bypass complicated by gastric pouch necrosis: considerations in gastroesophageal reconstruction.

Gastric pouch necrosis and intraabdominal sepsis is an uncommon complication following laparoscopic gastric bypass. The intraoperative management of this complication centers on resection of the necrotic pouch, esophageal diversion, drainage, and enteral access for nutrition. Reestablishing gastrointestinal continuity at a later surgery following this complication can be challenging. We present a case in which the colon was found to be unacceptable for use in reconstruction; the remaining stomach was used as the conduit for a transhiatal reconstruction of gastrointestinal continuity instead.