Differential pp60c-src activity in well and poorly differentiated human colon carcinomas and cell lines.

The results presented in this report demonstrate increased pp60c-src kinase activity associated with moderate to well differentiated colon tumors, corroborating previous observations by other groups. Extension of this analysis to include a small number of poorly differentiated colon carcinomas revealed src kinase activity comparable to that observed in normal colonic mucosa, considerably less than that observed in moderate/well differentiated lesions. Correlations of src kinase activity with differentiation was confirmed within a panel of colon cell lines where increased activity, associated with moderate/well differentiated lines, was accompanied by increased expression of pp60c-src protein. Use of an antiphosphotyrosine antibody in immunoprecipitation revealed the presence of novel phosphotyrosyl cellular substrates in human colon cell lines displaying elevated pp60c-src kinase activity. These observations suggest a role for the src protooncogene in colonic differentiation pathways.

Somatic frameshift mutations in DNA mismatch repair and proapoptosis genes in hereditary nonpolyposis colorectal cancer.

An exacerbated genomic instability at simple repeated sequences characterizes cancer of the microsatellite mutator phenotype (MMP). The majority of hereditary nonpolyposis colon cancers (HNPCCs) and about 15% of nonselected ("sporadic") gastrointestinal tumors belong to the MMP pathway of tumorigenesis. Colorectal MMP+ and MMP- tumors exhibit fundamental differences in genotype and phenotype. We have shown previously that "sporadic" MMP+ colon cancers exhibit a paradoxical low incidence of somatic mutations in the p53 tumor suppressor gene and the c-K-ras proto-oncogene. On the other hand, gastrointestinal MMP+ cancers frequently harbor frameshift mutations in genes containing mononucleotide repeats. These include the cell growth regulator gene TGFbetaRII and the proapoptotic gene BAX. We have also recently shown the frequent presence of frameshift mutations in (A)8 and (C)8 tracts within the hMSH3 and hMSH6 DNA mismatch repair genes in sporadic colon cancer of the MMP. Here, we describe the nearly identical incidence of somatic frameshift mutations in these genes in a panel of 27 HNPCC MMP+ cancers: 52% in hMSH3 and BAX and 33% in hMSH6. In contrast, no mutations in any of these genes were found in 10 MMP- cancers of HNPCC patients. These results show that the multistep model for the unfolding of the MMP also applies to HNPCC and further illustrate the importance of the escape from apoptosis in the MMP pathway for gastrointestinal cancer. They also underscore the differences in genotype between tumors with and without enhanced microsatellite instability and the similarities in genotype between tumors of the MMP regardless of their hereditary or sporadic nature.

An anoxia inducible endonuclease and enhanced DNA breakage as contributors to genomic instability in cancer.

Fischer rat embryo fibroblasts subjected to temporary anoxia followed by an aerobic recovery period show genomic instability in the form of highly elevated CAD gene amplification rates. As revealed by flow cytometric analysis this is associated with DNA breakage in vivo, followed by repair during the recovery period. Such genomic instability parallels expression of a M(r) 29,000/31,000 endonuclease; this enzyme requires no added divalent metal ion and has a pH optimum of about 6.5. The same endonuclease was found to be expressed within healing wounds and in four of ten human colorectal cancers but was not seen in eight normal colorectal tissue samples. Our results indicate that DNA breakage resulting from endogenous endonuclease activity can have a substantial effect in modulating genomic instability.

Genomic DNA-based hMSH2 and hMLH1 mutation screening in 32 Eastern United States hereditary nonpolyposis colorectal cancer pedigrees.

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant cancer syndrome characterized by early age of onset colorectal cancer (mean 45 years) as well as endometrial, urinary tract, and upper gastrointestinal adenocarcinomas. The HNPCC phenotype has been shown to segregate with germline mutations in the human homologues of the DNA mismatch repair genes MSH2, MLH1, PMS1, and PMS2. However, the majority of published DNA mismatch repair gene mutation surveys associated with HNPCC kindreds report multiple levels of preselection, including 2p and 3p chromosomal linkage analysis and the evaluation of microsatellite instability of proband colorectal cancers prior to mutation analysis. For this reason, the concise contribution of each of the known DNA mismatch repair genes to the HNPCC phenotype remains unknown. We report the results of a genomic DNA-based analysis of hMSH2 and hMLH1 germline mutations in 32 unrelated Eastern United States HNPCC kindreds. These families were selected for study on the basis of phenotype only. We identified three hMSH2 and six hMLH1 mutations in eight families, for a positive mutation rate of 25%. Two mutations were identified in one of the families. Four of the mutations detected have not been reported in the literature previously. One of the mutation-positive families is African American; the others were of European-American ancestry. These results provide a clarification of the contribution of hMSH2 and hMLH1 to the HNPCC phenotype and suggest that in the majority of Eastern United States HNPCC kindreds selected by phenotype alone, the molecular genetic basis for the disease remains unknown.

Absorbable mesh sling prevents radiation-induced bowel injury during "sandwich" chemoradiation for rectal cancer.

HYPOTHESIS: Absorbable mesh slings can prevent radiation-induced bowel injury when adjuvant pelvic radiotherapy is given in the early postoperative period. We hypothesized that the mesh sling technique is similarly effective during "sandwich" sequence adjuvant chemoradiation. DESIGN: Retrospective review. SETTING: Tertiary care comprehensive cancer center. PATIENTS: Nonrandomized series of 19 consecutive patients who underwent abdominoperineal resection and received postoperative sandwich sequence chemoradiation at Roswell Park Cancer Institute, Buffalo, NY, between January 1994 and September 1999. INTERVENTIONS: Twelve patients had an absorbable mesh sling placed at the completion of abdominoperineal resection. Seven patients did not have an absorbable mesh sling placed. MAIN OUTCOME MEASURES: Radiotherapy dose and gastrointestinal toxic effects. RESULTS: All 12 patients in the "mesh" group were able to receive full-dose radiotherapy with tumor bed boost (total dose, 54 Gy, 11 patients; 59.4 Gy, 1 patient). Only 3 of 7 patients in the "no mesh" group were able to receive a tumor bed boost (total dose, 46.8 Gy, 1 patient; 50.4 Gy, 3 patients; 54 Gy, 3 patients). Acute gastrointestinal toxic effects were minimal in the mesh group (grade 1, 10 patients; grade 2, 2 patients) compared with the no mesh group (grade 2, 6 patients; grade 3, 1 patients). None of the patients in the mesh group have shown evidence of late gastrointestinal toxic effects. One patient in the no mesh group required surgery for complications of chronic radiation enteritis. CONCLUSIONS: The protective effects of an absorbable mesh sling extend beyond the life expectancy of the mesh itself. Sandwich sequence chemoradiation should not preclude the use of the mesh sling technique.

The management of infectious and noninfectious anorectal complications in patients with leukemia.

BACKGROUND: Infectious and noninfectious anorectal complications may occur in patients undergoing therapy for leukemia. Controversy surrounds the treatment of this problem in immunocompromised patients. STUDY DESIGN: A retrospective review of the medical records of 83 patients with acute or chronic leukemia in whom anorectal disease developed during inpatient therapy for leukemia was performed to determine the initial signs and symptoms, treatment, and outcomes. RESULTS: During a 12-year period, 92 patients with anorectal complications were treated. This series included 25 patients with perirectal abscesses, 22 patients with anal fissures, 18 patients with symptomatic external hemorrhoids, 12 patients with perianal ulcerations, 12 patients with symptomatic internal hemorrhoids, and three patients with fistulas in ano. Overall, 79 (86 percent) of the 92 anorectal complications resolved in 68 of the 83 patients. Increasing periods of neutropenia did not adversely affect the resolution of anorectal disease. Thirteen patients (16 percent) required surgical intervention, most commonly secondary to a perirectal abscess. Incision and drainage was necessary in ten (40 percent) of 25 patients with perirectal abscess, which included five patients with fluctuation and five patients in whom infection failed to respond to medical therapy. CONCLUSIONS: Noninfectious anorectal complications in patients with leukemia respond to nonoperative intervention and rarely progress to a life-threatening infection. Nonoperative intervention in the form of systemic antibiotics and sitz baths is successful in the treatment of infectious anorectal complications. Incision and drainage should be performed when fluctuation is present and in patients whose complications fail to respond to medical therapy.

Colorectal cancer in patients aged 30 years or younger.

Colorectal cancer (CRC) is believed to carry a grim prognosis in young patients. A retrospective study of patients diagnosed with colorectal cancer at age 30 years or less between 1971 and 1994 was conducted. Statistical analyses were performed using non-parametric one way ANOVA tests and logistic regression models. Sixty-eight of the patients evaluated at our institution were suitable for this study. Risk factors were identified in 28% of patients. The median age at diagnosis was 27 years (range 14-30 years). Fifty-six patients (82%) were Stage III or IV at the time of diagnosis. Twenty-two of the 34 patients who underwent potentially curative surgery had recurring disease at a median of 12 months (range 1-43 months). At a median follow-up of 21.5 months, 54 patients had died from disease. At the time of death, abdominal carcinomatosis and distant disease were the most common patterns of failure. Stage of the primary tumour (P=0.0006) and recurrence (P=0.0001) were the only variables noted to be associated with survival. The stage of the primary tumour and whether the tumour recurred were each associated with survival in patients with colorectal cancer at age 30 years or less.

Utility of mapping lymph nodes cleared from rectal adenocarcinoma specimens.

The lymph node clearing technique improves the detection of lymph nodes in colorectal cancer specimens. The purpose of this study was to determine the utility of mapping the lymph nodes cleared from rectal adenocarcinoma specimens by evaluating the possible relationship between the pattern of lymph node metastases to the site of the recurrent disease. A retrospective medical record review was performed in 40 patients with primary rectal adenocarcinoma. The specimens were analysed by lymph node clearing technique and mapped after surgery. The lymph nodes were mapped according to their location in the cleared specimens. Statistical analysis was performed using the chi 2-test. A total of 1290 lymph nodes were cleared in 40 specimens. Of these, 1126 (87%) lymph nodes were < or = 5 mm. One-hundred and ten (9%) lymph nodes were metastatic. Sixty-seven (61%) of these 110 lymph nodes were 5 mm or less in size. The majority of lymph nodes with or without metastases were in the pelvis, as opposed to an extrapelvic location (P = 0.0001). Eleven patients recurred. In nine of these patients the recurrence showed a direct relationship between the level of metastatic lymph node location (pelvic vs. extrapelvic) and the site of the recurrent disease (loco-regional or systemic, P = 0.05). The majority of lymph nodes, both normal and metastatic, cleared from specimens from rectal adenocarcinoma were < or = 5 mm in diameter. The lymph node mapping technique may help in predicting the site of recurrence.

Sexual dysfunction, informed consent and multimodality therapy for rectal cancer.

BACKGROUND: This study assessed the presurgical and preradiation discussion of the risk of posttherapy sexual dysfunction among patients who underwent potentially curative therapy for rectal cancer. The incidence of sexual dysfunction after treatment for rectal cancer was then determined. METHODS: A retrospective review of the medical records of 52 consecutive patients who underwent potentially curative procedures for rectal cancer within 15 cm from the anal verge was performed. RESULTS: Presurgical discussion of the risk of sexual dysfunction was not documented in the consent in 37 of 52 patients (71%). Among the 5 males who underwent local excision, none reported posttherapy sexual dysfunction. Of the 6 males who were treated by low anterior resection, only 1 had a postoperative complaint of sexual dysfunction. Five of 15 males (33%) treated with abdominoperineal resection (APR) alone reported postprocedure sexual dysfunction, whereas 6 of 8 males (75%) treated with APR and radiation reported dysfunction. Of the entire female cohort, only 1 of the 16 reported sexual dysfunction posttherapy. CONCLUSION: A discussion of the risks of posttherapy sexual dysfunction was documented for fewer than one third of the patients. Among males after APR, the use of postoperative radiation showed a trend toward an increase in sexual dysfunction. Surgery and/or radiation therapy did not impact on sexual dysfunction in females.

Local excision for rectal cancer: an uncertain future.

Adenocarcinoma of the rectum remains a significant public health challenge, with 39,000 new cases and 8,500 deaths predicted for 1998. Radical surgery, the current standard therapy, frequently necessitates the formation of a permanent colostomy and is associated with significant morbidity. For these reasons, alternatives to radical surgery have been sought. This review focuses on sphincter-sparing surgical modalities for distal rectal cancer. An extensive review of the literature on local excision alone, local excision plus postoperative radiation therapy (with or without chemotherapy), and local excision following preoperative chemoradiotherapy is presented. The design and interim results of the sole prospective multi-institution trial of local excision, Cancer and Leukemia Group B trial 8984, are also summarized. The literature on this subject, which is dominated by single-institution, retrospective reports, fails to support local excision as a superior or equal therapy to radical surgical excision for invasive distal rectal adenocarcinoma. The crucial question regarding the efficacy of radical surgical salvage for local recurrence following local excision also remains unanswered. We conclude that the role of local excision for invasive distal rectal adenocarcinoma remains undefined. If there is a future for this therapeutic modality, it will depend significantly on rigorous patient selection, provided that the efficacy of radical surgical salvage for local recurrence can be established.

Impact of cooked functional meat enriched with omega-3 fatty acids and rosemary extract on inflammatory and oxidative status; a randomised, double-blind, crossover study.

BACKGROUND & AIM: n-3 fatty acid intake has been associated with inflammatory benefits in cardiovascular disease (CVD). Functionalising meat may be of great interest. The aim of the present study was to assess the effect of functional meat containing n-3 and rosemary extract on inflammatory and oxidative status markers in subjects with risk for CVD. METHODS AND RESULTS: A randomised, double-blind, cross-over study was undertaken to compare the effects on the above markers of consuming functional or control meat products. 43 volunteers with at least two lipid profile variables showing risk for CVD were randomly assigned to receive functional meat (FM) or control meat (CM) over 12-weeks with a 4-week wash-out interval before crossover. Functional effects were assessed by examining lipid profile, CRP, PAI-1, TNF-alpha, IL-6, fibrinogen (inflammatory markers), and TBARS, FRAP and 8-iso-PGF2 (oxidative status markers). 33 subjects (24 women) aged 50.7±8.8 years completed the study. In FM treatment, PAI-1, fibrinogen and 8-iso-PGF2 decreased significantly after 12 weeks, while FRAP significantly increased. In contrast, in CM treatment, a significant increase was seen in PAI-1, while FRAP significantly declined. Significant differences were also seen between the FM and CM treatments after 12 weeks in terms of the change observed in PAI-1, FRAP and 8-iso-PGF2 values. No significant differences were seen in anthropometric variables nor were adverse effects reported. CONCLUSION: The consumption of FM containing n-3 and rosemary extract improved oxidative and inflammatory status of people with at least two lipid profile variables showing risk for CVD. The inclusion of such functional meat in a balanced diet might be a healthy lifestyle option.

Objetivos: La ingesta de omega-3 se ha asociado con efectos antinflamatorios relacionados con la prevención de la enfermedad cardiovascular (ECV). Desarrollar productos cárnicos funcionales podría ser de gran interés para la población. El objetivo del presente estudio fue evaluar el efecto de una carne funcional con omega-3 y extracto de romero sobre marcadores de inflamación y oxidación en personas con riesgo cardiovascular. Pacientes y métodos: Se diseñó un ensayo clínico cruzado y doble-ciego para estudiar el efecto del consumo de un producto cárnico funcional sobre marcadores de inflamación y oxidación. Se incluyeron 43 voluntarios con al menos 2 parámetros del perfil lipídico alterado, indicando riesgo de ECV. Fueron asignados aleatoriamente en 2 grupos que consumieron en cruzado carne funcional (CF) o carne control (CC) durante 12 semanas con un periodo de lavado de 4 semanas entre ellos. Al finalizar el estudio se evaluó: perfil lipídico, marcadores de inflamación (PCR, PAI-1, TNF-alpha, IL-6, fibrinógeno) y marcadores de oxidación (TBARS, FRAP, 8-iso-PGF2). Resultados: Completaron el estudio 33 personas (24 mujeres) con edad media de 50.7±8.8 años. Tras consumir CF durante 12 semanas se observó una disminución significativa del PAI-1, fibrinógeno y 8-iso-PGF2, mientras que el FRAP incrementó significativamente. Sin embargo, con CC incrementó PAI-1 y disminuyó FRAP significativamente. Además se observaron diferencias significativas entre los cambios producidos tras consumir uno u otro producto de los marcadores PAI-1, FRAP y 8-iso-PGF2. Al final de cada intervención no se observaron cambios en variables antropométricas ni efectos adversos. Conclusiones: El consumo de CF con omega-3 y extracto de romero mejora el estado inflamatorio y oxidativo de personas con al menos 2 parámetros del perfil lipídico alterado. La inclusión de estas CF en una dieta equilibrada podría ser una opción más para mantener un estilo de vida saludable. ClinicalTrials.gov NCT0199088.

Utility of routine postoperative laboratory studies in patients undergoing potentially curative resection for adenocarcinoma of the colon and rectum.

In an effort to lower healthcare costs, this study was undertaken to evaluate the utility of routine postoperative (PO) laboratory studies and determine whether abnormalities alter patient (PT) care. This was a retrospective review of 105 PTs undergoing elective curative resection for colorectal cancer. A serum electrolyte and liver panel and a hematologic panel were drawn in all PTs. OF 8749 total laboratory values obtained, 5894 (67%) were normal. Two of these (0.03%) elicited a therapeutic intervention. Of the 2004 values that were low (23%), 103 (5.1%) elicited a therapeutic response. Of the 851 that were high (10%), 21 (2.5%) elicited a therapeutic response. Of 2089 preoperative laboratory values, 252 (12%) were abnormal, but in only 15 incidences in 9 PTs was any action taken. Three PTs required potassium supplementation and 6 PTs were transfused packed red blood cells before surgery. In the PO period 2603 laboratory values of 6660 obtained (39%) were abnormal. Of these, 735 (28%) were high and 1868 (72%) were low. Twenty of 735 (27%) high values triggered a therapeutic response that most commonly required administration of insulin for elevated serum glucose in 17 of 197 occasions in five diabetic PTs. On three occasions potassium was removed from intravenous fluids. Five of 275 (1.8%) low calcium values were treated in five patients. Potassium was replaced in 17 of 32 occasions in 14 patients where it was low. In this group of PTs, PO serum potassium, hemoglobin levels, and serum glucose in diabetics were the only values important in making therapeutic decisions. If laboratory studies can be streamlined into only those necessary, substantial savings in health care will be seen without sacrificing quality medical care.

Adenosquamous carcinoma of the colon and rectum.

PURPOSE: Adenosquamous carcinomas of the colon and rectum are rare tumors. These tumors have malignant glandular and squamous components capable of metastasizing. We report seven patients with adenosquamous carcinoma of the colon and rectum and their clinical outcomes. METHODS: Retrospective review was undertaken of seven patients who were identified by the tumor registry at Roswell Park Cancer Institute between 1971 and 1994. RESULTS: These represent a 0.18 percent incidence of adenocarcinomas in our institution. There were five tumors located in the rectum and two in the colon. All patients were Stage III or IV on presentation. Median overall survival was 23 months. In three patients, the tumor was associated with ulcerative colitis; in one of these patients, hypercalcemia was present. All patients died of their disease or with the disease. CONCLUSIONS: Previously reported aggressive behavior of this cancer is confirmed in our series.

Clinical implications of multiple colorectal carcinomas in hereditary nonpolyposis colorectal carcinoma.

PURPOSE: An increased incidence of multiple (synchronous and metachronous) colorectal carcinomas has been reported in hereditary nonpolyposis colorectal cancer. This review was undertaken to determine the clinical implications of multiple colorectal carcinomas in hereditary nonpolyposis colorectal cancer. METHODS: A retrospective review of the records of patients in the hereditary nonpolyposis colorectal cancer registry at Roswell Park Cancer Institute who had either synchronous or metachronous colorectal carcinomas was conducted. RESULTS: Twenty-five of 93 patients with documented pathology were found to have multiple colorectal carcinomas. The mean age at diagnosis of the index colorectal carcinoma was 46.7 (range, 28-65) years. There were 7 (7.5 percent) patients with synchronous colorectal carcinomas and 20 (21.5 percent) patients with metachronous colorectal carcinomas. Two of the seven (28.6 percent) patients with synchronous colorectal carcinomas developed a metachronous colorectal carcinoma. In the patients with metachronous colorectal carcinomas, 29 metachronous events were noted: colon (23) and rectum (6). The mean and median time interval for metachronous colorectal carcinomas were 10.9 and 11.8 (range, 1.5-43.8) years, respectively. The mean times to first, second, and third events were 11.7 (range, 1.5-43.5), 7.9 (range, 2.7-18.7), and 12.3 (range, 11.8-12.7) years, respectively. The majority of patients with metachronous colorectal carcinomas did not have stage progression at the diagnosis of the metachronous colorectal carcinomas: 13 patients had lower or same stage at first event, 4 had lower or same stage at second event, and 2 patients had lower stage at third event. Three of 20 patients with metachronous colorectal carcinomas died of their disease. CONCLUSION: Multiple colorectal cancers are common in hereditary nonpolyposis colorectal cancer. Even though stage progression may not be evident at diagnosis of metachronous colorectal cancer, some of these patients will nevertheless die of their disease.

Modulation of mdr-1 expression by a H-ras oncogene in a human colon carcinoma cell line.

In an established colon differentiation model, introduction of a c-H-ras-1 oncogene into a poorly differentiated human colon carcinoma cell line (Clone A) results in changes associated with the acquisition of a more differentiated phenotype. Down-regulation of mdr-1 mRNA was shown to accompany ras-related differentiation events resulting in decreased Pgp synthesis and a significant reduction in membrane Pgp as detected by immunoprecipitation, Western-blot and FACS analysis. Consistent with these observations was a reduction in Pgp-mediated drug resistance associated with Clone-A ras transfectants, with no alteration in drug sensitivity being observed with non-MDR drugs in these cells. An alternative differentiation model involves exposure of Clone-A cells to sodium butyrate. Under these conditions, differentiation-related changes resulted in up-regulation of mdr-1 mRNA and Pgp synthesis, although no alteration in drug sensitivity was recorded. In agreement with this observation, the levels of membrane-associated Pgp remained unchanged throughout the period of exposure to sodium butyrate. This study shows that modulation of Pgp expression in colon differentiation is dependent upon the differentiation induction agent used.

Constitutive expression of multidrug resistance in human colorectal tumours and cell lines.

In this study we report detection of mdr1 gene expression in the liver metastases of 7/11 patients with colon carcinoma and characterise the MDR phenotype associated with a panel of 19 human colon carcinoma cell lines. Within this panel, mdr1 mRNA biosynthesis and surface localisation of Pgp were assessed with respect to MDR functionality where the cell lines are representative of different clinical stages of tumour progression, metastatic potential and differentiation. The data indicates that constitutive levels of mdr1 mRNA/Pgp expression may not necessarily result in the functional expression of the MDR phenotype. While low levels of mdr1 mRNA/Pgp were detected in 5/8 well differentiated colon cell lines, only 2/8 were functionally MDR. In contrast, 10/11 moderate and poorly differentiated lines expressed mdr1 mRNA/Pgp and of these, 9/11 were functionally MDR. The phosphorylation status of the mature 170 kD P-glycoprotein and the surface localisation of this glycoprotein showed the strongest correlation with functionality. Analysis of cell lines for cross-resistance and chemosensitivity profiles against a battery of chemotherapeutic drugs suggests multiple mechanisms, in addition to Pgp, contribute to the overall resistance of colorectal cancer.

Photodynamic therapy for residual neoplasms of the perianal skin.

PURPOSE: The aim of this study was to evaluate the efficacy of photodynamic therapy in the management of residual neoplasms of the perianal skin. METHODS: This is a retrospective review. Five patients with pathologic confirmation of residual perianal neoplasms were treated with photodynamic therapy. There were three females. The mean age was 52 (range, 33-79) years. Pathology consisted of Bowen's disease in two patients, squamous-cell carcinoma in two patients, and extramammary Paget's disease in one patient. Four patients received one photodynamic therapy treatment and one patient received two treatments three months apart. RESULTS: Treatment was followed by immediate perianal erythema, subsequent blister formation in 36 to 48 hours, and sloughing of the treated area in 72 hours. With a mean follow-up of 5.2 (range, 1-8) years, there were two recurrences. One recurrence was in a patient four years after treatment for Paget's disease, and the other was in a patient nine months after treatment for Bowen's disease. The latter was managed successfully with wide local excision. Treatment-related toxicities included significant perianal pain in four patients, controlled with analgesia management. CONCLUSIONS: Photodynamic therapy can successfully be used after wide local excision for residual neoplasms of the perianal skin. Treatment can be rendered with acceptable morbidity.

Inhibition of N-linked glycosylation of P-glycoprotein by tunicamycin results in a reduced multidrug resistance phenotype.

Characterisation of altered glycosylation of P-glycoprotein (P-gp) found associated with the absence of a multidrug resistance (MDR) phenotype in cell lines prompted an investigation to assess the role of post-translational processing in establishing P-gp efflux pump functionally. The clone A cell line used in this study displays a strong MDR phenotype mediated by high constitutive levels of expression of P-gp. Incubation of clone A cells with tunicamycin for different periods resulted in a time-dependent increase in daunorubicin accumulation, reflecting a reduction in P-gp function. Parallel experiments conducted with verapamil resulted in no loss of P-gp functionality in clone A cells. Reduction in surface-associated P-gp following exposure to tunicamycin was established by FACS analysis, Western blot analysis and immunoprecipitation of surface-iodinated P-gp. In addition, immunoprecipitation of P-gp from 32P-orthophosphate-labelled cells demonstrated reduced phosphorylation of P-gp associated with tunicamycin exposure. From these studies we conclude that glycosylation of P-gp is required to establish the cellular MDR phenotype.