Nutrient stress alters the glycosylation status of LGR5 resulting in reduced protein stability and membrane localisation in colorectal tumour cells: implications for targeting cancer stem cells.

BACKGROUND: LGR5 is an important marker of intestinal stem cells and performs its vital functions at the cell membrane. Despite the importance of LGR5 to both normal and cancer stem cell biology, it is not known how microenvironmental stress affects the expression and subcellular distribution of the protein. METHODS: Nutrient stress was induced through glucose starvation. Glycosylation status was assessed using endoglycosidase or tunicamycin treatment. Flow cytometry and confocal microscopy were used to assess subcellular distribution of LGR5. RESULTS: Glucose deprivation altered the glycosylation status of LGR5 resulting in reduced protein stability and cell surface expression. Furthermore, inhibiting LGR5 glycosylation resulted in depleted surface expression and reduced localisation in the cis-Golgi network. CONCLUSIONS: Nutrient stress within a tumour microenvironment has the capacity to alter LGR5 protein stability and membrane localisation through modulation of LGR5 glycosylation status. As LGR5 surface localisation is required for enhanced Wnt signalling, this is the first report to show a mechanism by which the microenvironment could affect LGR5 function.

Heterocyclic amines and genotype of N-acetyltransferases as risk factors for prostate cancer.

A variety of carcinogenic heterocyclic amines are produced during the cooking of meat at high temperatures. These carcinogens are metabolized by N-acetyltransferases (NAT), which are polymorphic in the population. This study examined associations between prostate cancer (PCa) and the consumption of different kinds of meat, heterocyclic amine intake and NAT genotypes. PCa patients and controls were recruited in the Syracuse, NY area. Levels of meat and heterocyclic amine intakes were determined from validated surveys and NAT genotypes were determined by the sequences of PCR-amplified DNA from buccal swabs. A total of 152 cases and 161 controls were eligible for analysis. There was an association between PCa and history of PCa in the first-degree blood relatives (OR = 4.59, 95% CI 2.21-9.70), and family history of bladder cancer (P < 0.02). However, there was no association with the history of other cancers. There was no association between PCa and either 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) intake, or NAT1 and NAT2 genotypes. However, there was a trend of association with MeIQx and with rapid NAT2 and NAT1*10 in combination with PhIP. A new NAT1 allele with a frequency of one out of 544 chromosomes was found in the Caucasian subjects.

The present and future of appointment, tenure, and compensation policies for medical school clinical faculty.

The authors present data and information about appointment, tenure, and compensation policies to describe how medical schools are redefining the terms under which they relate to their full-time clinical faculties. First, the authors note the increasing differentiation of clinical faculty members into two groups, researchers and clinicians. The present-day competitive realities of both research and clinical enterprises have prompted this change and the principles of mission-based management are reinforcing it. Second, they document the long-term tendency of schools to appoint new clinical faculty members to contract-term (as opposed to tenure) appointments, as special non-tenure-eligible tracks for clinically oriented faculty proliferate. Third, they report on the policies of schools to limit the financial guarantees provided to clinical faculty members who are awarded tenure. For schools that have yet to address this issue, they discuss the various employment and pay arrangements that inform or confuse the question. Fourth, they describe historic problems with clinical faculty compensation arrangements and illustrate, with examples from ten schools, the characteristics of recently implemented performance- and risk-based compensation plans. While these trends in institutional policies and practices may initially concern faculty advocate groups, the authors argue that they may serve the long-term interests of those groups. The terms of relationships between medical schools and their clinical faculties are tied closely to the specifics of organizational structure, which are currently undergoing review and change. The challenge all schools face is to define these terms in ways that allow them to continue to attract high-quality clinical faculty while avoiding an insupportable financial liability.

Organizational models for medical school-clinical enterprise relationships.

Changes in the organization, financing, and delivery of health care services have prompted medical school leaders to search for new organizational models for linking medical schools, faculty practice groups, affiliated hospitals, and insurers-models that better meet the contemporary challenges of governance and decision making in academic medicine. However, medical school leaders have relatively little information about the range of organizational models that could be adopted, the extent to which particular organizational models are actually used, the conditions under which different organizational models are appropriate, and the ramifications of different organizational models for the academic mission. In this article, the authors offer a typology of eight organizational models that medical school leaders might use to understand and manage their relationships with physicians, hospitals, and other components of clinical delivery systems needed to support and fulfill the academic mission. In addition to illustrating the models with specific examples from the field, the authors speculate about their prevalence, the conditions that favor one over another, and the benefits and drawbacks of each for medical schools. To conclude, they discuss how medical school and clinical enterprise leaders could use the organizational typology to help them develop strategy and manage relationships with each other and their other partners.

Murine animal model.

Experimental animal models are available for the development of new treatment. Murine animal models have particular advantages for comparative study to evaluate the efficacy and safety of different treatment modalities because many mice can be treated at the same time with easy handling. Among several experimental models, murine renal carcinoma (Renca), which arises spontaneously in Balb/c mice, is the most frequently used for the assessment of chemotherapy, immunotherapy, and radiotherapy. Renca cells readily establish tumors in isogenic mice, producing histologically proven adenocarcinoma with a predictable growth rate to mimic the clinical situation for orthotopic growth and metastasis in a reasonable time frame. Because of its poor immunogenicity and its responsiveness to immunotherapy, the number of studies using cytokine gene-modified tumor vaccines-such as interferon-alpha or interleukin-2-in the Renca system is growing. Therefore, Renca experiments greatly contribute to the analysis of the mechanisms of antitumor immune response. In this chapter, we describe several experimental systems using this Renca model.

Measuring contributions to the clinical mission of medical schools and teaching hospitals.

This is the final report of a panel convened as part of the Association of American Medical College's (AAMC's) Mission-based Management Program to examine the use of metrics (i.e., measures) in assessing faculty and departmental contributions to the clinical mission. The authors begin by focusing on methods employed to estimate clinical effort and calculate a "clinical full-time equivalent," a prerequisite to comparing productivity among faculty members and departments. They then identify commonly used metrics, including relative-value units, total patient-care gross charges, total net patient fee-for-service revenue, total volume per CPT (current procedural terminologies) code by service category and number of patients per physician, discussing their advantages and disadvantages. These measures reflect the "twin pillars" of measurement criteria, those based on financial or revenue information, and those based on measured activity. In addition, the authors urge that the assessment of quality of care become more highly developed and integrated into an institution's measurement criteria. The authors acknowledge the various ways users of clinical metrics can develop standards against which to benchmark performance. They identify organizations that are sources of information about external national standards, acknowledge various factors that confound the interpretation of productivity data, and urge schools to identify and measure secondary service indicators to assist with interpretation and provide a fuller picture of performance. Finally, they discuss other, non-patient-care, activities that contribute to the clinical mission, information about which should be incorporated into the overall assessment. In summary, the authors encourage the use of clinical productivity metrics as an integral part of a comprehensive evaluation process based upon clearly articulated and agreed-upon goals and objectives. When carefully designed, these measurement systems can provide critical information that will enable institutional leaders to recognize and reward faculty and departmental performance in fulfillment of the clinical mission.

Review of US medical school finances, 1998-1999.

Based on data from the Annual Medical School Questionnaire of the Liaison Committee on Medical Education, to which 100% of the 125 accredited allopathic US medical schools responded, we found that revenue supporting programs and activities of the 125 accredited medical schools in the United States totaled $39,761 million in 1998-1999. Three sources accounted for 79.3% of total revenues: practice plans ($13,724 million; 34.5%), grants and contracts ($11, 982 million; 30.1%), and hospital support ($5814 million; 14.6%). In the aggregate, total revenues increased by 7.4% between 1997-1998 and 1998-1999, a consequence at least in part due to a 2.9% increase in the number of full-time faculty. The largest increase in dollar amount came from grants and contracts ($1101 million; 10.2% increase). Revenue increases were not evenly distributed across the schools. Increases of 10% or more in key revenue sources-practice plans and hospital support-were reported by approximately one fourth of all schools. Another one fourth reported decreases in these same sources. JAMA. 2000;284:1127-1129

Trends in US medical school faculty salaries, 1988-1989 to 1998-1999.

Expansion of managed care, intensified price competition, and the introduction of the Medicare Fee Schedule have all affected physician compensation during the past decade. We examine trends in the salaries of medical school faculty, particularly MD clinical faculty, based on a more extensive salary database than has been used previously. Data collected through the Association of American Medical Colleges' Faculty Salary Survey for the academic years 1988-1989, 1993-1994, and 1998-1999 were analyzed, and inflation-adjusted salary growth rates for clinical and basic science faculty during two 5-year periods, 1988-1993 and 1993-1998, compared across faculty ranks, departments, and various school characteristics. The comparison showed that, between 1988 and 1998, the actual median clinical faculty salary increased from $101,000 to $150,000, and the actual median basic science faculty salary increased from $52,000 to $78,000. Bivariate and multivariate analyses showed that the proportionate change in real mean salary (base year, 1988) in each 5-year period was related to department and faculty rank for clinical faculty (P<.001) and faculty rank for basic science faculty (P<.001). The inflation-adjusted annualized compound growth rate of clinical faculty salaries declined from 1.9% per year (1988-1993) to 0.2% per year (1993-1998), while the growth rate of basic science faculty salaries increased from 0.3% per year (1988-1993) to 1.3% per year (1993-1998). From 1993 to 1998, inflation-adjusted annualized salary growth rates in several clinical departments were negative (anesthesiology, -1.1%; obstetrics and gynecology, -0.5%; radiology, -0.4%; and neurology, -0.1%) but were positive for family practice (+2.7%). Significant differences in salary growth related to school characteristics (eg, geographic region, public vs private, community based vs non-community based, and research intensity) were specific to particular study periods. Overall, while actual average medical school faculty salaries are increasing, the real growth rate of average clinical faculty salaries is declining and that of basic science faculty increasing. JAMA. 2000;284:1130-1135

Carcinogenicity of the N-hydroxy derivative of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 2-amino-3, 8-dimethyl-imidazo[4,5-f]quinoxaline and 3, 2'-dimethyl-4-aminobiphenyl in the rat.

Heterocyclic amines and carcinogenic aromatic amines are similarly metabolically activated suggesting that they may have similar organ specificity. Three day-old male ACI/seg rats were injected, i.p., twice a week for 10 weeks with 50 micromol/kg of N-hydroxy-3, 2'-dimethyl-4-aminobiphenyl (N-OH-DMABP; Group II), N-OH-2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline (N-OH-MeIQx; Group III) or N-OH-2-amino-1-methyl-6-phenylimidaza-[4,5-b]pyridine (N-OH-PhIP; Group IV). Animals in control group (Group I) were similarly injected with solvent alone. The animals were sacrificed at age 68 weeks, and 31, 30, 27 and 31 rats from Groups I, II, III and IV, respectively, were evaluated. Colon carcinomas were found in 0, 15 (P<0.001), 2 and 4 (P<0.06), and bladder transitional cell tumors in zero, two, two and four (P<0.06), in Group I, II, III and IV, respectively. The incidence of atypical hyperplasia of ventral prostate in Groups III and IV, and of anterior prostate and seminal vesicle in all treated groups was also significantly greater (P<0.05). These results suggest that N-OH-PhIP and N-OH-MeIQx may be potential carcinogens for the prostate. Since bladder tumor is rare in ACI rats, N-OH-PhIP may also be a potential carcinogen for the bladder.

Measuring contributions to the research mission of medical schools.

The authors of this article, who were the members and staff of a research panel formed by the AAMC as part of its mission-based management initiative, reflect on the growing interest in quantitative information in the management of the research mission of medical schools. They note the serious limitations of any such system of measures for research, particularly its inability to represent directly the quality of the research effort. Despite these concerns, the authors acknowledge that leaders in academic medicine have always used quantitative measures in one form or another to compare performance or assess progress. Two factors appear to be driving increases in this practice: (1) the need to demonstrate to institutional stakeholders that resources are being used wisely and that the school's performance justifies continued investment in the research mission; and (2) the need to fashion an economic strategy to manage precious institutional resources, particularly research space. Given these realities, the authors offer guidelines for the proper development and use of measures to assess contributions by faculty, departments, and institutions to the research mission. They also comment on the measures most commonly used in four areas: grants and other revenue-generating activities; publications; faculty members' research reputation and contributions to the national research enterprise; and support to the general research mission of the school. The authors conclude that quantitative information can help institutional leaders in important management decisions. However, the potential for misuse is great. The key is always to regard this information as an aid to judgment, not a substitute for it.

Inhibition of lung metastases of murine renal cell carcinoma by the combination of radiation and interferon-alpha-producing tumor cell vaccine.

We have previously demonstrated in a murine lung metastasis model that local sublethal radiation of tumors can synergistically enhance their sensitivity to immunotherapy with either systemic high-dose interleukin-2 (IL-2) or vaccination with autologous tumor cells expressing IL-2, interferon (IFN)-gamma and granulocyte-macrophage colony-stimulating factor (GM-CSF). Host antitumor activity was mediated in large part by natural killer cells, which can be activated by IFN-alpha. In the present study, we used this lung metastasis model to investigate the efficacy of combined therapy with local tumor radiation and vaccination with IFN-alpha-secreting tumor cells (Renca/IFN-alpha). The in vitro and in vivo growth rates of Renca/IFN-alpha cells were significantly reduced relative to normal controls. Subcutaneous vaccination with Renca/IFN-alpha or selective X-irradiation of the left lung (300 rad) reduced the number of lung tumors by 40% and 27%, respectively. The combination of lung irradiation plus vaccination reduced the number of lung metastases by 60%, and the net tumor volume by 95%. The reductions in tumor volume in both irradiated and non-irradiated lungs were comparable. These results indicate that host antitumor response to subcutaneous vaccination with Renca/IFN-alpha was systemic, and was significantly enhanced by radiation of tumor-bearing lungs. A regimen based on enhancement of IFN-alpha immunotherapy by local tumor radiation may be useful in the treatment of metastatic renal cell carcinoma.

Adding value to security services.

The need of the security manager to market himself or herself and the value of the services he or she performs has become a critical element of security operations today in all industries, including health care. In this article, the author reviews the wide range of options to be considered in offering security services both inside and outside your facility.

Review of US medical school finances, 1997-1998.

Based on data from the Annual Medical School Questionnaire of the Liaison Committee on Medical Education (LCME), to which 100% of schools responded, we found that revenue supporting programs and activities of the 125 accredited medical schools in the United States totaled $36997 million in 1997-1998. A large proportion of revenue (79%) was derived from 3 sources: practice plans ($12559 million; 33.9%), grants and contracts ($10916 million; 29.5%), and hospital support ($5741 million; 15.5%). An analysis of revenue trends revealed that medical schools, in aggregate, have continued to experience growth during the last 2 years. However, the aggregate numbers mask considerable variation among schools with regard to changes in financing. Between 1995-1996 and 1996-1997, 46 schools (37%) reported constant-dollar declines in the sum of practice plan and hospital revenue, and 50 schools (40%) reported a decline from 1996-1997 to 1997-1998. The financial data reviewed in this report demonstrate the continued dependence of medical schools on faculty-generated sources of revenue and confirm the perception that a growing number of medical schools are experiencing reductions in key sources of financial support. Current and projected reductions in teaching hospital revenue due to the implementation of the Balanced Budget Amendment are expected to erode further hospital support for medical school programs and activities.

N-Acetyltransferase expression and DNA binding of N-hydroxyheterocyclic amines in human prostate epithelium.

Intact prostate epithelial cells prepared from benign prostatic hypertrophy tissues from two patients were incubated for 2 h with N-hydroxy derivatives of 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (N-OH-PhIP) or 2-amino-3,8-dimethylimidazo[4, 5-f]quinoxaline (N-OH-MeIQx). (32)P-post-labeling analysis detected PhIP and MeIQx adducts in the DNA of these cells but not in the untreated control. Adduct levels were approximately 100 times greater in N-OH-PhIP- than in N-OH-MeIQx-treated cells. Repair synthesis of DNA was observed in cells, prepared from two additional patients, treated for 24 h with these carcinogens and was greater for N-OH-PhIP than for N-OH-MeIQx. PhIP, MeIQx and their nitro derivatives did not produce repair synthesis of DNA in this system. The difference in the activity of N-OH-PhIP and N-OH-MeIQx may be due to their stability, since N-OH-MeIQx decomposed rapidly in neutral solution. Transcripts of NAT1 and NAT2 were detected by an in situ hybridization method in prostate epithelial cells, but were absent from stromal tissues. These results suggest that PhIP may be a potential carcinogen for human prostate, since cooked meats, which contain this heterocyclic amine, have been associated with human prostate cancer.

Immunotherapy for lung metastases of murine renal cell carcinoma: synergy between radiation and cytokine-producing tumor vaccines.

We investigated the combination therapy of local radiation of lung metastasis and vaccination with autologous tumor cells that produced interleukin (IL)-2, interferon-gamma (IFN-gamma), and granulocyte-macrophage colony-stimulating factor (GM-CSF) using the mouse Renca pulmonary metastasis model. Wild-type Renca (W/Renca) were transfected with pEF-BOS vector incorporating cDNAs for IL-2, IFN-gamma, or GM-CSF to express these cytokines. W/Renca, IL-2-producing Renca (Renca/IL-2), and IFN-gamma-producing Renca (Renca/IFN-gamma) produced subcutaneous tumor at the injection site in eight of eight, one of eight, and two of eight mice, respectively. No tumors were found in the GM-CSF-producing Renca (Renca/GM-CSF) group (zero of eight). Renca/IFN-gamma produced subcutaneous (s.c.) tumors in all Balb/c nude mice, but Renca/IL-2 and Renca/GM-CSF did not. To test the elicitation of antitumor activity, Balb/c mice were injected intravenously with 1 x 10(5) W/Renca on day 0, vaccinated, s.c., with 1 x 10(6) cells each of 5,000 rad preirradiated Renca/IL-2, Renca/IFN-gamma, and Renca/GM-CSF or 3 x 10(6) cells of preirradiated W/Renca on days 1, 7, and 14, and radiated with 300 rad to both lungs on day 5. The animals were killed on day 21 and tumor nodules in the lungs were enumerated. Neither local irradiation alone nor the combination of lung radiation and multiple vaccination with irradiated W/Renca significantly reduced the number of lung tumors. In contrast, the combination of lung radiation and the multiple vaccinations with cytokine-producing Renca cells significantly reduced the number of lung tumors. This regimen was more effective than the multiple vaccinations with cytokine-producing Renca cells alone. These studies demonstrate the efficacy of vaccination with autologous tumor cells expressing these cytokines and sensitization of the tumor target with radiation.

Protection against mammary tumor growth by vaccination with full-length, modified human ErbB-2 DNA.

ErbB-2 is overexpressed in several human cancers and conveys a transforming activity that is dependent on tyrosine kinase activity. Antibodies and T cells to ErbB-2 have been isolated from cancer patients, indicating ErbB-2 as a potential target of active vaccination. In this study, 3 mutant ErbB-2 DNA constructs encoding full-length, ErbB-2 proteins were tested as tumor vaccines. To eliminate tyrosine kinase activity, the ATP binding lysine residue 753 was substituted with alanine by replacing codon AAA with GCA in mutant ErbB-2A. To direct recombinant ErbB-2 to the cytoplasm where major histocompatibility complex (MHC) I peptide processing takes place, the endoplasmic reticulum (ER) signal sequence was deleted in cyt ErbB-2. The third construct cyt ErbB-2A contained cytoplasmic ErbB-2 with the K to A mutation. Expression of recombinant proteins was measured by flow cytometry in transfected murine mammary tumor cell line D2F2. Transmembrane ErbB-2 and ErbB-2A were readily detected. Cytoplasmic ErbB-2 and ErbB-2A were detected only after the transfected cells were incubated overnight with a proteasome inhibitor, indicating prompt degradation upon synthesis. ErbB-2 autophosphorylation was eliminated by the K to A mutation as demonstrated by Western blot analysis. Growth of ErbB-2-positive tumor in BALB/c mice was inhibited after vaccination with ErbB-2 or ErbB-2A, but not with cyt ErbB-2 or cyt ErbB-2A. ErbB-2A that is free of tyrosine kinase activity is a potential candidate for anticancer vaccination. The 3 mutant constructs should be useful tools to delineate the role of individual immune effector cell in ErbB-2-specific antitumor immunity and to develop strategies for enhancing such immunity.

Expression of p53 and pRb in bladder and prostate cancers of patients having both cancers.

Evidence has been presented that tumor suppressor genes p53 and Rb play a crucial role in the development of both human prostate and bladder cancer. Patients with either cancer are at an increased risk for developing the other malignancy as compared to the general population. The purpose of the present study was to investigate whether there is abnormal expression of these two suppressor proteins in both the bladder and prostate cancers of the same patient. The expression of p53 and pRb in bladder and prostate cancer specimens obtained from 15 patients having both cancers was studied using immunohistochemical staining with antibodies against these proteins. The expression of p53 and pRb in both bladder and prostate cancers of the same patient was congruent in 8 of 15 cases (53%) for p53 and 9 of 15 cases (60%) for pRb. The significance of these findings warrants further investigations.

Review of US medical school finances, 1996-1997.

Based on data from the Annual Medical School Questionnaire of the Liaison Committee on Medical Education, to which 100% of schools responded, the revenues that supported the programs and activities of the 125 accredited medical schools in the United States totaled $34897 million in 1996-1997. A large proportion (78.9%) of these revenues was derived from 3 sources: practice plans, grants and contracts, and hospital support. Both public and private medical schools, in aggregate, have continued to experience growth throughout the last decade but at a progressively slower rate, primarily because of a slowing in the growth of practice plan revenues. Federal revenues supporting research in public and private medical schools since 1992-1993 have grown at annualized, constant-dollar rates of 5.6% and 4%, respectively. Growth in state and local appropriations to public medical schools has tended to lag behind inflation. Growth in reported revenues from endowments that are used to support programs at private medical schools is on the rise. The aggregate numbers mask considerable variation among schools with regard to changes in financing. A small, but appreciable, number of schools have witnessed a constant-dollar decline in their total practice plan revenues since 1992-1993. The financial data reviewed in this report demonstrate the continued dependence of medical schools on faculty-generated sources of revenue and confirm the perception that medical schools, as a group, are experiencing constraints on the growth of their enterprises.

Neoplastic progression of breast epithelial cells--a molecular analysis.

Molecular changes associated with breast cancer progression were characterized using the MCF-10F cell series. MCF-10F was established from fibrous mastectomy tissue of a patient without detectable cancer. In vitro treatment of MCF-10F cells with benzo(a)pyrene resulted in a transformed subclone MCF-10F-BP1 (BP1). Transfection of clone BP1 with T24-Hras resulted in the tumorigenic line MCF-10F-BP1-Tras (BP1-Tras). Using flow cytometry, the expression of HLA I, ERBB-2 and MUC-1 was found to be comparable in 'normal' MCF-10F, transformed BP1 and tumorigenic BP1-Tras cells. Glycosylated mucin is elevated in BP1 but reduced in BP1-Tras cells. Using mRNA differential display analysis, cDNA profiles of the 'normal', transformed and tumorigenic cell lines were strikingly similar, yet distinct and elevated expression of several common cDNA fragments was detected in BP1 and BP1-Tras when compared with MCF-10F cells. These fragments were cloned and sequenced. The sequences of clones T1-360 and C4-310 are homologous to two reported EST cDNA clones from human fetal tissue and were further characterized. Elevated expression of the genes corresponding to clones T1-360 and C4-310 was verified using Northern blotting. High-level expression of these genes was also detected in the breast cancer cell line MCF-7 that was derived from the pleural effusion of a patient with advanced breast cancer. Therefore, specific molecular changes associated with breast cancer development were identified and may be indicators of neoplastic progression.