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J L Mulshine

Publications and source records attributed to J L Mulshine.

At least 37 records · Page 2Linked to original sources
PubMed1999

Autocrine growth loops dependent on peptidyl alpha-amidating enzyme as targets for novel tumor cell growth inhibitors.

Many small cell lung tumors are dependent in vitro and in vivo on autocrine growth loops. The prototypical small cell lung cancer autocrine growth factor, gastrin-releasing peptide (GRP), is one of many peptide hormones which require post-translational carboxy-terminal alpha-amidation for bioactivity. We have reported that neuroendocrine human lung tumor cell lines express the bifunctional enzyme PAM which catalyzes the biosynthesis of alpha-amidated peptides in a two-step process, and have recently shown that non-small cell lung cancer cell lines and tumors, generally considered to be non-endocrine in nature, also express PAM. We have also shown that two chemical classes of PAM inhibitors, substrate analogues and specific copper chelators, inhibit amidating enzyme activity in cell-free extracts. Here we demonstrate in vitro growth inhibition of lung cancer tumor cell lines by both these classes of PAM inhibitors using the MTT assay and the clonogenic assay. Growth inhibition in a small cell lung cancer cell line can be overcome by exogenous addition of synthetic alpha-amidated GRP. Similar growth-suppressive effects are seen in cell lines stably transfected with a vector expressing antisense PAM RNA. These data support the mechanism of inhibition for a new type of chemotherapeutic/intervention agent, directed at synthesis and activation of peptide growth factors, and support our postulate that alpha-amidated peptide hormones are a common component in lung tumor autocrine growth biology which can be inhibited by targeting the biochemical mechanisms necessary for growth factor synthesis.

Anti-Bacterial Agents↗
PubMed1999

Reducing lung cancer risk: early detection.

Strategies for the early detection of lung cancer are being investigated in an attempt to improve the poor prognosis associated with the disease. Such approaches, which include the identification of biomarkers for preclinical disease, must be integrated into multimodal cancer prevention strategies. Recent investigations have identified potential markers of early disease, including heterogeneous nuclear ribonucleoprotein, although the use of multiple markers may be required to provide the sensitivity and specificity necessary for mass screening. Early detection necessitates the development of effective chemoprevention strategies for the airway-confined phase of lung cancer. Current research efforts explore the utility of direct drug delivery, such as with the use of aerosolized delivery of retinoids, to maximize delivery of the active agent to the site of early lung cancer while avoiding systemic adverse effects.

Aerosols↗
PubMed1999

Phase I and imaging trial of a monoclonal antibody directed against gastrin-releasing peptide in patients with lung cancer.

Small cell lung cancer (SCLC) cells express and secrete bombesin-like peptides (BLP) that can activate specific receptors that stimulate the growth of these cells. A murine monoclonal antibody, 2A11, which binds to the BLP, gastrin-releasing peptide with high affinity, has been reported to decrease the growth of SCLC cells in vitro and in athymic nude mice. A Phase I trial in lung cancer patients was performed using multiple doses of 2A11. Thirteen patients with lung cancer received 12 doses of 2A11 antibody three times a week for 4 weeks at one of four dose levels. Serum samples were obtained prior to initiation and before each dose of 2A11 antibody therapy for measurement of 2A11 antibody levels and determination of serum human anti-mouse antibody levels. A pilot imaging evaluation using 111In conjugated 2A11 monoclonal antibody was also performed in the same patients to aid in the study of pharmacokinetics and biodistribution. No toxic reactions were observed, and none of the patients developed detectable human antimouse antibody; however, no objective antitumor responses were observed. The mean trough serum 2A11 levels in patients increased with increasing dose level: 0.26+/-0.2 microg/ml, 6.7+/-6 microg/ml, 71.5+/-60 microg/ml, 248+/-184 microg/ml for dose levels 1 mg/m2, 10 mg/m2, 100 mg/m2, and 250 mg/m2, respectively. At each dose level, sustained detectable serum levels of the monoclonal antibody were achieved. Tumor uptake was noted in 11 of 12 patients who were injected with 111In conjugated 2A11. Because no dose-limiting clinical toxicity was observed, a mathematical model was used to define the recommended Phase II dose of 250 mg/m2. This trial established that repeated doses of monoclonal antibody 2A11 could be given safely to patients, and sustained levels could be achieved for a 4-week schedule. Further evaluation of the antitumor effects of 2A11 is warranted.

Animals↗
PubMed1998

Expression of heterogeneous nuclear ribonucleoprotein A2/B1 changes with critical stages of mammalian lung development.

Recent reports have demostrated a link between expression of members of the family of heterogeneous nuclear ribonucleoproteins (hnRNPs) and cancer. Overexpression of hnRNP A2/B1 correlated with the eventual development of lung cancer in three different clinical cohorts. We have studied the expression of hnRNP A2/B1 messenger RNA (mRNA) and protein during mammalian development. The expression of hnRNP A2/B1 mRNA and protein are parallel but change dynamically during critical periods in mouse pulmonary development. hnRNP A2/B1 is first detected in the lung in the early pseudoglandular period, peaks at the beginning of the canalicular period, and remains high during the saccular (alveolar) period. In mouse and rat, hnRNP A2/B1 expression is first evident in the earliest lung buds. As lung development progresses, the cuboidal epithelial cells of the distal primitive alveoli show high levels of the ribonucleoprotein, which is almost undetectable in the proximal conducting airways. The expression of hnRNP A2/ B1 is restricted in mature lung. Similar dynamic pattern of expression through lung development was also found in rat and human lung. Upregulated expression of hnRNP A2/B1 at critical periods of lung development was comparable to the level of expression found in lung cancers and preneoplastic lesions and is consistent with hnRNP A2/B1 overexpression playing an oncodevelopmental role.

Animals↗
PubMed1998

Expression of heterogeneous nuclear ribonucleoprotein A2/B1 in bronchial epithelium of chronic smokers.

The monoclonal antibody 703D4, which binds heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1), has been reported to detect lung cancer more than a year earlier than routine chest X-ray or cytomorphology. To explore the biological basis of this detection, we studied the expression of this antigen in the central airways of smokers with evidence of bronchial metaplasia using specimens from a previously reported, randomized retinoid chemoprevention trial. By analyzing 1078 available biopsy specimens from 147 individuals at baseline and 68 individuals who completed the intervention, we frequently detected overexpression of hnRNP A2/B1 in normal and abnormal bronchial epithelium (i.e., in 41% of normal and 37% of squamous metaplasia samples). There was no correlation between hnRNP A2/B1 overexpression and the different histological changes. In cases with hnRNP A2/B1 overexpression, immunoreactivity was homogeneously expressed in all biopsied sites. For the 68 cases with serial biopsies, there was no significant modulation of hnRNP expression by retinoid intervention or smoking status. With lung cancer cell lines, 0.5-4 microM concentrations of 13-cis-retinoic acid reduced hnRNP A2/B1 overexpression by immunocytochemistry. We conclude that hnRNP A2/B1 overexpression is frequently found in central airways of chronic smokers, consistent with the pattern of expression that we reported previously in airways surrounding resected primary lung cancers. Oral 13-cis-retinoic acid at a dose of 1 mg/kg has no demonstrable effects on modulating hnRNP A2/B1 expression in proximal bronchial epithelium.

Biomarkers, Tumor↗
PubMed1998

Phase I study of the pharmacokinetics of a radioimmunoconjugate, 90Y-T101, in patients with CD5-expressing leukemia and lymphoma.

Ten patients with advanced or refractory CD5-expressing hematologic neoplasms [two with chronic lymphocytic leukemia and eight with cutaneous T-cell lymphoma (CTCL)] were treated in a Phase I study with the radioimmunoconjugate 90Y-T101, which targets CD5+ lymphocytes. Prior imaging studies using 111In-T101 demonstrated uptake in involved lymph nodes and skin in patients with CTCL, and Phase I studies with unmodified T101 demonstrated transient responses. In this study, patients were treated with 5 or 10 mCi of 90Y chelated to T101 via isothiocyanatobenzyl diethylenetriamine pentaacetic acid, along with tracer doses of 111In-T101 for imaging. The biodistribution of the radioimmunoconjugate was determined by measuring 90Y and 111In blood clearance, urine excretion, and accumulation in bone marrow and in involved skin lesions. The intravascular pharmacokinetics of 90Y were predicted by 111In-labeled T101. The greatest differences in biodistribution between 111In and 90Y were in the higher bone accumulation of 90Y and its lower urinary excretion. Imaging studies demonstrated targeting of skin lesions and involved lymph nodes in CTCL patients. The predominant toxicity was bone marrow suppression. Rapid antigenic modulation of CD5 on circulating T and B cells was observed. Recovery of T-cell populations occurred within 2-3 weeks; however, suppression of B-cell populations persisted after 5+ weeks. All CTCL patients developed human antimouse antibody after one cycle and thus were not retreated; one patient with chronic lymphocytic leukemia received a second cycle of therapy. Partial responses occurred in five patients, two with chronic lymphocytic leukemia and three with CTCL. The median response duration was 23 weeks. One CTCL patient who subsequently received electron beam irradiation to a residual lesion is disease-free after 6 years.

Adult↗
PubMed1997

New approaches to the integrated management of early lung cancer.

We have briefly surveyed some the developments in the field of molecular diagnostics that provide a basis for cautious optimism about progress in population-based early lung cancer screening. The sound lung cancer management strategies that were formulated several decades ago failed in clinical trials because the necessary tools to implement the strategies were not yet available. Technology is beginning to emerge that makes population-based screening achievable. This same technology may be used to define a comprehensive marker panel including the most informative markers from the long list of candidate markers. Validation studies will define more clearly the strengths and limitations of new molecular diagnostics and provide leads for further research attention. The clinical community can expedite this process if these validation efforts are aggressively pursued. Parallel developments are clearly needed in refining the range of therapeutic intervention for early cancer management. The success of both diagnostic and intervention tools is interwoven in the ultimate goal of reducing lung cancer mortality. This article is an invitation to think expansively about new approaches to cancer care that integrate the fruits of our hard-learned lessons.

Humans↗
PubMed1997

Recent molecular advances in the approach to early lung cancer detection and intervention.

Lung cancer is a major contributor to overall cancer mortality. Detecting lung cancer while it is still a localized process is a long-cherished goal for improving the outcome of this disease. Recent developments suggest that we are approaching this capability. We next have to think about how to implement a change in our approach to lung cancer management to derive the benefit of better detection capability. This is an area in which our growing understanding of lung cancer biology is providing clues on improving the inhibition of cancer progression.

Apoptosis↗
PubMed1997

Antitumor activity of a monoclonal antibody directed against gastrin-releasing peptide in patients with small cell lung cancer.

BACKGROUND: Small cell lung cancer (SCLC) cells express and secrete gastrin-releasing peptide (GRP) which binds to receptors and stimulates growth of these cells. A murine monoclonal antibody, 2A11, which binds GRP with high affinity, decreased growth of SCLC cells in vitro and in athymic nude mice. A phase 1 trial and pharmacokinetic modeling in patients with lung cancer has defined the phase 2 dose of 2A11 but the antitumor activity in patients is unknown. METHODS: Thirteen patients with previously treated SCLC received 2A11 at 250 mg/m2 over 1 h three times per week for 4 weeks. Serum GRP, urine GRP, serum levels of 2A11, and human antimouse antibodies (HAMA) were determined. RESULTS: One of 12 (8%; 95% confidence interval, 0 to 38%) evaluable patients had complete resolution of radiographically detectable tumor lasting 4 months. Four patients (33%) had stable disease. No toxic reactions were observed. The pretreatment serum GRP level of the responding patient was 3.1 fmol/mL and the median of nine nonresponding patients was 7.3 fmol/mL (range, <1.0 to 29.0). The mean trough serum 2A11 level was 49+/-18 microg/mL in the responding patient and 32 to 487 mg/mL (median, 117) in 10 nonresponding patients. HAMA did not increase during 2A11 administration in any patient. CONCLUSIONS: Interruption of the GRP autocrine growth factor loop with 2A11 results in clinical antitumor activity in a minority of patients with previously treated SCLC. Further evaluation of the antitumor effects of 2A11 is warranted to define characteristics associated with response to 2A11.

Animals↗
PubMed1997

Prospective detection of preclinical lung cancer: results from two studies of heterogeneous nuclear ribonucleoprotein A2/B1 overexpression.

The United States lung cancer epidemic has not yet been controlled by present prevention and treatment strategies. Overexpression of a Mr 31,000 protein, heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1, had shown promise as a marker of lung cancer. In a pilot study of archived preneoplastic sputum specimens, hnRNP A2/B1 overexpression more accurately detected preclinical lung cancer than standard cytomorphology. In separate, ongoing prospective studies, sputum is collected annually from stage I resected non-small cell lung cancer patients at high risk of developing a second primary lung cancer and Yunnan tin miners at high risk of primary lung cancer. After the first year of follow-up, preclinical detection of lung cancer by routine cytology was compared with hnRNP A2/B1 overexpression as measured by quantitative densitometry of immunostained slides. Up-regulation of hnRNP A2/B1 in sputum specimens accurately predicted the outcome in 32 of 40 primary lung cancer and control patients within 12 months, whereas cytological change suggestive of lung cancer was found in only 1 patient. In the primary lung cancer study, overexpressed hnRNP A2/B1 accurately predicted the outcome in 69 of 94 primary lung cancer and control miners, whereas only 10 with primary lung cancer were diagnosed cytologically. These two prospective studies accurately predicted that 67 and 69% of those with hnRNP A2/B1 up-regulation in their sputum would develop lung cancer in the first year of follow-up, compared with background lung cancer risks of 2.2 and 0.9% (35- and 76-fold increase, respectively). Using sputum cells to monitor hnRNP A2/B1 expression may greatly improve the accuracy of preclinical lung cancer detection.

Aged↗
PubMed1997

High frequency of K-ras codon 12 mutations in bronchoalveolar lavage fluid of patients at high risk for second primary lung cancer.

A high frequency of K-ras mutations may indicate preneoplastic changes in the bronchial epithelium as a result of genotoxic injury. With the use of sensitive detection techniques, we report a higher prevalence of K-ras mutations in bronchoalveolar lavage than has been reported previously for lung cancer. A PCR/ligase chain reaction technique was used to determine K-ras codon 12 mutations in a group of 52 bronchoalveolar lavage specimens from patients at risk of a second lung cancer. Of the specimens examined, 84% contained at least one mutation in K-ras codon 12, corroborated by an allele-specific hybridization method. These results suggest that point mutations in K-ras codon 12 are widespread in the bronchial epithelium. Based on these preliminary findings, further evaluation of this efficient sensitive assay to monitor K-ras status should be conducted in larger clinical cohorts where clinical outcomes will ultimately be available. Such a trial will define the utility of K-ras codon 12 mutation status as a marker of lung cancer.

Bronchoalveolar Lavage Fluid↗
PubMed1996

Detection of K-ras oncogene mutations by polymerase chain reaction-based ligase chain reaction.

To evaluate a rapid multiplexed assay to detect three common K-ras codon 12 mutations, primer pairs complementary to the wild-type and mutant loci were developed and tested with lung cancer cell lines with previously identified mutation status. The sensitivity of detection of mutations was determined to be at least 1% using spiked samples containing K-ras codon 12 mutations. This assay was then used to evaluate prospectively K-ras status in airways of individuals at high risk of lung cancer by analysis of bronchoalveolar lavage (BAL) specimens from patients who have been previously treated for lung cancer. DNA was extracted from BAL specimen cell pellets, and PCR-based ligase chain reaction was performed for mutations in the first position of codon 12 of K-ras, with positive and negative controls. Of 10 BAL samples, 4 contained 1 mutation (GGT --> TGT), 1 contained 2 mutations (GGT --> TGT and GGT --> AGT), and the rest were wild-type. The BAL mutations were validated by cloning and screening with mutant-specific probes followed by confirmation sequencing.

Bronchoalveolar Lavage↗
PubMed1996

Insulin-like growth factor expression in human cancer cell lines.

The insulin-like growth factors (IGFs), IGF-I and IGF-II, are potent mitogens for human lung and other epithelial cancer cell lines. Previous studies in defined medium lacking added IGF or insulin suggest that an IGF-related ligand can act as an autocrine growth factor for many cancer cell lines through action via the type I IGF receptor (IGF-R). Analysis of RNA isolated from human lung and breast cancer cell lines by reverse transcription of mRNA and polymerase chain reaction reveal that IGF-I and IGF-II mRNAs were co-expressed with IGF-R in the majority of cell lines. IGF-I mRNA was detected in 11/12 small cell lung cancer cell lines (SCLC), 13/14 nonsmall cell lung cancer (NSCLC) cell lines, and 1/2 breast cancer cell lines. IGF-II mRNA was detected in 8/10 SCLC, 11/12 NSCLC cell lines, and 2/2 breast lines. All cell lines expressed IGF-R. For analysis of IGF peptide secretion, cell lines were adapted to growth in serum/hormone-free culture medium (R0), and to avoid interference by IGF-binding proteins, secreted IGF peptides were isolated under acidic conditions and analyzed by Western blotting. Based upon measurement of the sensitivity of the anti-IGF antibodies for detection of recombinant human IGFs, IGF peptides accumulated in conditioned medium at greater than picomolar concentrations should have been readily detected. In three cell lines (two lung and one breast) secreted IGF immunoreactivity was detected as three molecular mass species of 23, 14, and 6 kDa. Isolation and NH2-terminal sequencing of each of these species definitively identified them as differentially processed forms of the IGF-II prohormone. Despite the high frequency of IGF-I gene expression detected by reverse transcription-polymerase chain reaction analysis, only one lung cancer cell line, NCI-N417d, was found that unequivocally secreted IGF-I peptide. This direct sequence determination unambiguously identifies IGF-II as the predominant IGF involved in the autocrine growth stimulation of human lung and breast epithelial tumor cell lines and supports a growing body of literature that implicates IGF-II/IGF-R autocrine loops as a common growth mechanism in epithelial carcinogenesis.

Adenocarcinoma↗
PubMed1996

Purification and characterization of a protein that permits early detection of lung cancer. Identification of heterogeneous nuclear ribonucleoprotein-A2/B1 as the antigen for monoclonal antibody 703D4.

We have reported that a mouse monoclonal antibody 703D4, detects lung cancer 2 years earlier than routine chest x-ray or cytomorphology. We purified the 703D4 antigen to elucidate its role in early lung cancer biology, using Western blot detection after SDS-polyacrylamide gel electrophoresis. Purification steps included anion exchange chromatography, preparative isoelectric focusing, polymer-based C18-like, and analytical C4 reverse phase high performance liquid chromatography. After 25-50,000-fold purification, the principal immunostaining protein was > 95% pure by Coomassie staining. The NH2 terminus was blocked, so CNBr digestion was used to generate internal peptides. Three sequences, including one across a site of alternate exon splicing, all identified a single protein, heterogeneous nuclear ribonucleoprotein-A2 (hnRNP-A2). A minor co-purifying immunoreactive protein resolved at the final C4 high performance liquid chromatography step is the splice variant hnRNP-B1. Northern analysis of RNA from primary normal bronchial epithelial cells demonstrated a low level of hnRNP-A2/B1 expression, consistent with immunohistochemical staining of clinical samples, and increased hnRNP-A2/B1 expression was found in lung cancer cells. hnRNP-A2/B1 expression is under proliferation-dependent control in normal bronchial epithelial cell primary cultures, but not in SV40-transformed bronchial epithelial cells or tumor cell lines. With our clinical data, this information suggests that hnRNP-A2/B1 is an early marker of lung epithelial transformation and carcinogenesis.

Amino Acid Sequence↗
PubMed1996

Growth control of lung cancer by interruption of 5-lipoxygenase-mediated growth factor signaling.

Signal transduction pathways shared by different autocrine growth factors may provide an efficient approach to accomplish clinically significant control of lung cancer growth. In this study, we demonstrate that two autocrine growth factors activate 5-lipoxygenase action of the arachidonic acid metabolic pathway in lung cancer cell lines. Both growth factors increased the production of 5(S)-hydrooxyeicosa-6E,8Z,11Z,14Z-tetraeno ic acid (5-HETE), a major early 5-lipoxygenase metabolic product. Exogenously added 5-HETE stimulated lung cancer cell growth in vitro. Inhibition of 5-lipoxygenase metabolism by selective antagonists resulted in significant growth reduction for a number of lung cancer cell lines. Primary clinical specimens and lung cancer cell lines express the message for the 5-lipoxygenase enzymes responsible for the generation of active metabolites. In vivo evaluation demonstrated that interruption of 5-lipoxygenase signaling resulted in enhanced levels of programmed cell death. These findings demonstrate that 5-lipoxygenase activation is involved with growth factor-mediated growth stimulation for lung cancer cell lines. Pharmacological intervention with lipoxygenase inhibitors may be an important new clinical strategy to regulate growth factor-dependent stages of lung carcinogenesis.

5-Lipoxygenase-Activating Proteins↗
PubMed1996

NCI-Navy Medical Oncology Branch cell line data base.

The cell line data base described in this paper includes both clinical information about the patients from whom the cell lines were derived and information about the in vitro analyses performed of the cell lines. The cell line data base has evolved as a part of a systematic effort by a research group at the NCI since 1976 to generate human cell lines as biological tools to study cancer and other diseases. The cell lines were generated from clinical specimens obtained as part of a series of Institutional Review Board-approved clinical protocols. The preponderance of the data is on lung cancer cell lines, though a broad range of other cancers are represented. A bank of over 300 human cell lines including cancer cell and in some instances autologous B-lymphoblastoid cells from the NCI-VA and NCI-Navy Medical Oncology Branch are reposited at the American Type Culture Collection. The cell lines are available for the research community. The entire data base is available on the American Type Culture Collection Web Site (WWW:http:@www.atcc.org/).

Biological Specimen Banks↗