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Biomedical subjects

R H Whitehead

Publications and source records attributed to R H Whitehead.

At least 55 records · Page 3Linked to original sources
PubMed1990

Development of a database of amino acid sequences for human colon carcinoma proteins separated by two-dimensional polyacrylamide gel electrophoresis.

The tandem use of preparative two-dimensional polyacrylamide gel electrophoresis (2-DE) and electroblotting onto polyvinylidene difluoride membranes has been employed to rapidly isolate a number of proteins from a crude cell extract of a human colon carcinoma cell line (LIM 1863). The immobilized proteins were located by staining with Coomassie Brilliant Blue R-250, and selected protein spots were excised and subjected to Edman degradation. Our results demonstrate that overall sequence yields in the 3-20 pmol range can be achieved on protein spots from four identical 2-DE gels; approximately 150-200 micrograms of total protein was applied to a single 2-DE gel. An approximate two-fold increase in sensitivity of phenylthiohydantoin-amino acid detection (subpicomole range) was achieved by fitting our commercial sequencers with a simple sample transfer device which permitted the analysis of the total phenylthiohydantoin-amino acid derivative. N-Terminal amino acid sequence data was obtained for thirteen electroblotted proteins. All of these sequences positively matched those of proteins of known structure listed in the available protein sequence databases. Approximately 40% of the electroblotted proteins did not yield N-terminal sequence information, presumably because they had blocked N-termini (either naturally or artifactually). Internal amino acid sequence information was obtained from three proteins isolated by preparative 2-DE. This was achieved by in situ digestion of the proteins in the gel matrix with Staphylococcus aureus V8 protease, electrophoresis of the generated peptides in a one-dimensional gel, electrotransfer of the peptides to a polyvinylidene difluoride membrane and microsequence analysis of the electroblotted peptides.

Amino Acid Sequence↗
PubMed1990

Changes in the DNA ploidy patterns of human colorectal carcinomas, subsequent to culture or xenografting.

DNA ploidy patterns were measured by flow cytometry in 7 human carcinomas and compared with the DNA histograms of 3 cell lines and 25 xenografts and derived from them. Four of the carcinomas showed DNA ploidy changes on initial xenografting or early passaging, while 2 carcinomas showed stable ploidy patterns over several xenograft passages. One xenograft derived from a cell line showed a marked, but stable, increase in DNA content over the original human carcinoma. Two of the 3 cell lines were DNA aneuploid, and at least 1 line was apparently derived from a minor subpopulation in the original carcinoma. The possible mechanisms and implications of these changes are discussed.

Animals↗
PubMed1990

Intravenous but not intracolonic epidermal growth factor maintains colonocyte proliferation in defunctioned rat colorectum.

The role of epidermal growth factor in the proliferation of normal and premalignant colonocytes in vivo is not fully understood. In particular, the relative importance of its possible systemic and intraluminal routes of action has not been fully clarified. Rats with surgically defunctioned distal colorectums were used, and mini osmotic pumps were implanted to study the effects of intraluminal and IV administration of epidermal growth factor on colonocyte proliferation. Within 2 weeks of bypass, colonocyte proliferation in defunctioned colorectum has decreased to about one third the rate in normal colorectum or in colorectum proximal to the defunctioning colostomy. Intraluminal epidermal growth factor, infused from the time of operation did not reverse this hypoplasia, whereas IV epidermal growth factor maintained colonocyte proliferation at approximately the normal rate in bypassed colorectum. This model is suitable for testing other putative colonic mitogens for possible intraluminal and systemic effects.

Animals↗
PubMed1990

Autocrine regulation of human tumor cell proliferation by insulin-like growth factor II: an in-vitro model.

We have shown that a pleomorphic cell line of abnormal human karyotype derived from a stomach carcinoma (LIM-1839) proliferates in serum-free medium, expresses insulin-like growth factor II (IGF-II) mRNA, and secretes IGF-II (up to 56 ng/ml in serum-free conditioned medium, as measured in a rat liver RRA. No detectable levels of IGF-I can be measured in serum-free conditioned medium by RIA. These cells also secrete IGF-binding proteins, detected by a charcoal adsorption assay. The release of IGF-II and IGF binding proteins into serum-free conditioned medium (1.7 pmol/10(6) cells.24 h and 0.8 pmol binding sites/10(6) cells.24 h for 3 days, respectively) is inhibited 80% by cycloheximide (10 micrograms/ml). The LIM-1839 cells have type I and type II IGF receptors, determined by affinity cross-linking and competition binding studies. These cells proliferated 1.6-fold over 4 days in serum-free medium, with fresh medium changes on days 0 and 2: their growth was inhibited 56% by 40 micrograms/ml Sm 1.2, a monoclonal antibody which recognizes IGF-I and IGF-II. The addition of 20 and 50 ng/ml multiplication stimulating activity (rat IGF-II) caused 1.8- and 1.7-fold increases in cell growth between days 0 and 4 compared to controls, while [Thr59]IGF-I, at 20 and 50 ng/ml, caused 1.6- and 2.0-fold increases. Insulin, at 2 and 10 micrograms/ml, had no significant effect. The stimulatory effects of endogenous and exogenous IGFs on LIM-1839 cell proliferation were inhibited by a monoclonal antibody to the type I IGF receptor, alpha IR-3. These results suggest that the LIM-1839 cells are biologically responsive to endogenously produced IGF-II, and may thereby provide an in vitro model for autocrine regulation of human tumor growth by IGF-II.

Antibodies, Monoclonal↗
PubMed1989

A new gastric carcinoma cell line (LIM1839) derived from a young Caucasian male.

We describe a new and unique gastric carcinoma cell line (LIM1839) derived from a young Caucasian male with rapidly progressing disease. The cell line grows with a pleomorphic morphology and has been in continuous culture for more than 3 years. The cells cannot be cloned in semi-solid agar or grown in nude mice despite numerous attempts. The karyotype of the cultured cells is highly abnormal with a large number of structural and numerical changes. Some chromosomes are dicentric and this feature has persisted in this culture. The cells express one of the small-intestinal dipeptidases, aminopeptidase N, but do not express dipeptidyl peptidase IV or the disaccharidases, sucrase isomaltase or maltase glucoamylase. The cells express high levels of EGF receptors and of messenger RNA for insulin-like growth factor II.

Adenocarcinoma↗
PubMed1988

A technique for the in vitro culture of human parathyroid gland tissue.

A technique for prolonged in vitro culture of human parathyroid tissue is described. Cells could be maintained in this monolayer system and were shown to continue releasing high levels of parathyroid hormone into their culture medium even after 140 days in culture. Furthermore, growth of fibroblasts, persistence of epithelial cells and parathyroid hormone release was demonstrated in cells derived from parathyroid tissue that had been cryopreserved for 2 years. The availability of viable and functioning human parathyroid tissue produced in this culture system may be of value in both auto- and allo-transplantation in patients with permanent hypoparathyroidism.

Culture Techniques↗
PubMed1988

Tuft (caveolated) cells in two human colon carcinoma cell lines.

The presence of an unusual cell type in two human colon carcinoma cell lines is reported. The cells show the same morphology as "tuft" (caveolated) cells present in normal gastrointestinal epithelium. Tuft cells were seen in cell line LIM 1863 growing in vitro and in human colon carcinoma cell line LIM 2210 growing as subcutaneous solid tumour xenografts in nude mice. Characteristic morphologic features of tuft cells included a wide base, narrow apex and a tuft of long microvilli projecting from the apical surface. The microvilli are attached by a core of long microfilaments passing deep into the apical cytoplasm. Between the microvilli are parallel arrays of vesicles (caveoli) containing flocculent material. Two different but not mutually exclusive explanations for the presence of tuft cells are proposed. The first explanation is that tuft cells came from the resected tumour and have survived by mitotic division during subsequent passages. The second explanation suggests that tuft cells are the progeny of undifferentiated tumour cells. Descriptions of tuft cells in colon carcinomas are uncommon and possible reasons for this are presented. The morphology of tuft cells is consistent with that of a highly differentiated cell specialised for absorption, and these new models provide an opportunity to further investigate the structure and function of tuft cells.

Carcinoma↗
PubMed1987

Epidermal growth factor stimulates the synthesis of cell-attachment proteins in the human breast cancer cell line PMC42.

The human breast cancer cell line PMC42 responds to the addition of epidermal growth factor (EGF) by proliferation and increased frequency of attachment of cell-organoid structures to the culture vessel. Antibodies to fibronectin and laminin reacted strongly, by immunoperoxidase, with the membranes of cells from organoids that became adherent following addition of EGF. This reaction was weak with membranes of cells of non-adherent organoids in cultures containing EGF and was negative with membranes of cells of free-floating organoids from cultures without EGF. An increase in biosynthetic labelling with 35S-methionine was found in cell lysates and supernatants of PMC42 cells cultured in the presence of EGF compared with control cells grown without EGF. One-dimensional SDS-polyacrylamide gel electrophoresis and 2-dimensional NEPHGE-PAGE of labelled cell proteins showed increased synthesis of several cellular proteins and the appearance in EGF-treated cells of 2 proteins which were not detected in cells from control cultures lacking EGF. Immunoprecipitation experiments using antibodies to fibronectin and laminin with lysates of 35S-methionine labelled PMC42 cells cultured with EGF showed strong immunoprecipitation at Mr 200 and 400 with anti-laminin, and at Mr 200 and 96 with anti-fibronectin. These immunoprecipitates were blocked specifically by purified laminin or fibronectin, respectively. No immunoprecipitates were detected with these antibodies in lysates from cells grown without EGF. EGF thus stimulates increased adherence of cultured PMC42 cell-organoid structures together with increased membrane expression of the cell-adhesive proteins laminin and fibronectin. These effects may play a role in normal development and neoplastic behaviour of breast epithelia.

Breast Neoplasms↗
PubMed1987

A new colon carcinoma cell line (LIM1863) that grows as organoids with spontaneous differentiation into crypt-like structures in vitro.

A new colon carcinoma cell line (LIM1863) has been characterized. This cell line is unique in that the culture consists of organoids which are morphologically and functionally organized. Histological studies of the organoids show that the cells are arranged around a central lumen and the nuclei are polarized to the periphery. Two major morphological types are present: a columnar cell with a polarized, structurally normal brush border and goblet cells. The cells are also functionally mature and express the brush border enzymes aminopeptidase N, dipeptidyl peptidase IV, alkaline phosphatase, and sucrase-isomaltase. These enzymes are localized to the luminal membrane and the apical cytoplasm (of some cells). The goblet cells contain mucus and this mucus is secreted into the lumen. This functional differentiation suggests that the organoids contain precursor cells capable of differentiating along both the columnar and goblet cell pathways. At present no endocrine cells have been detected by morphological or histochemical analysis. The organoids have been in continuous culture with regular passaging for 21 months and also grow and differentiate normally in serum-free medium.

Aged↗
PubMed1987

A method for the isolation and culture of human colonic crypts in collagen gels.

Little is known concerning the biological factors that control the proliferation of the stem cells of the colonic mucosa. In part this is due to a lack of systems suitable for studying the proliferation of this mucosa in vitro. We describe a simple technique for the isolation of single viable intact crypts which are free of stroma and which can then be cultured for periods of at least 16 d using a collagen gel culture method. This method of crypt isolation was efficient with the mean yield of viable intact crypts being 1.4 +/- 1.2 X 10(4) (mean +/- 1 SD) crypts/cm2 of mucosa. In culture, mucosal cells only survived for extended periods when the crypts were cultured in collagen gels over a feeder layer of bovine aortic endothelial cells. Cells containing mucus were present in the cultured crypts at all stages of the culture; however we have not been able to demonstrate alkaline phosphatase activity in these crypts. Studies of DNA synthesis after 7 d in culture, using a 18-h pulse label with bromodeoxyuridine (BUdR) has shown that DNA synthesis, as measured by incorporation of BUdR into nuclei, is still occurring in these cultured crypts.

Cell Separation↗
PubMed1987

Single chromosome defect, partial trisomy 1q, in a colon cancer cell line.

The characterization of a single chromosome defect previously reported in a case of inherited nonpolyposis colorectal cancer is the essence of this communication. The defect, originally described as 13p+, is now being defined and the karyotype designated as 46,XY,-13,+der(13)t(1;13)(q32.1;p11).

Cell Line↗
PubMed1987

Chromosome studies in inherited nonpolyposis colon cancer syndrome.

This study was designed to determine if any constitutional chromosomal markers were linked with the expression of colorectal neoplasms in the inherited nonpolyposis colon cancer syndrome, using a number of cytogenetic techniques. High resolution G-banding in 12 affected and 17 unaffected family members did not reveal a structural chromosome abnormality. Increased C-band heteromorphism was not seen in either affected or unaffected individuals, and no heritable fragile sites were detected. Mean baseline and mitomycin C-induced sister chromatid exchanges were not elevated in affected patients compared with controls. Mapping of sister chromatid exchanges did not reveal any hot spots of exchange. A tumor cell line with the karyotype 46,XY,der(13),t(13;?)(p11;?) was established from one patient, but no constitutional abnormality of chromosome #13 was found. In addition, 11 patients with familial polyposis coli were studied with high resolution G-banding and no heteromorphism of chromosome #2 in the region 2q21.3 was detected.

Cell Line↗
PubMed1986

A monoclonal antibody CIBr17 recognizes a myoepithelium-specific antigen in human mammary gland.

A murine monoclonal antibody (MAb) CIBr17, raised against the human breast carcinoma cell line PMC42, reacts specifically with myoepithelial cells in normal human breast. This IgGl antibody recognizes a approximately 110kDa glycoprotein that is expressed on the cell surface and junctional membranes of PMC42 monolayer cultures. The CIBr17 antigen is present in two major glycosylated forms with approximate pls of 5.2 and 5.5 respectively in PMC42 cells. The tissue specificity of CIBr17 was assessed on frozen sections of PLP-fixed tissues by means of a 4-layer immunoperoxidase technique. CIBr17 has reacted with a variety of epithelium-derived tissues and some smooth muscle cells. Within many epithelial tissues, CIBr17 has demonstrated specific staining of particular epithelial cell types. Within normal breast and most benign breast lesions, antibody CIBr17 stained only myoepithelial cells. No staining of luminal epithelium, basement membranes or stromal elements was observed. In sclerosing adenosis, CIBr17 stained areas of pronounced myoepithelial differentiation, while in duct epitheliosis variable staining of proliferating cells was observed. In breast carcinomas, CIBr17 demonstrated variable antigen expression. In most tumors, CIBr17 either did not stain any tumor cells or stained only a small number of tumor cells spread randomly throughout the tumor. In several ductal carcinomas, however, CIBr17 stained the majority of tumor cells.

Antibodies, Monoclonal↗
PubMed1986

Binding of fluoresceinated epidermal growth factor to A431 cell sub-populations studied using a model-independent analysis of flow cytometric fluorescence data.

A method is developed for determining ligand-cell association parameters from a model-free analysis of data obtained with a flow cytometer. The method requires measurement of the average fluorescence per cell as a function of ligand and cell concentration. The analysis is applied to data obtained for the binding of fluoresceinated epidermal growth factor to a human epidermoid carcinoma cell line, A431. The results indicate that the growth factor binds to two classes of sites on A431 cells: 4 X 10(4) sites with a dissociation constant (KD) of less than or equal to 20 pM, and 1.5 X 10(6) sites with a KD of 3.7 nM. A derived plot of the average fluorescence per cell versus the average number of bound ligands per cell is used to construct binding isotherms for four sub-populations of A431 cells fractionated on the basis of low-angle light scatter. The four sub-populations bind the ligand with equal affinity but differ substantially in terms of the number of binding sites per cell. We also use this new analysis to critically evaluate the use of 'Fluorotrol' as a calibration standard in flow cytometry.

Carcinoma, Squamous Cell↗
PubMed1986

Long-term (5-11 years) follow-up of general immune competence in breast cancer. I. Pre-treatment levels with reference to micrometastasis.

General immune competence was measured before treatment in 185 breast cancer patients. They were then followed for 5 to 11 years to determine its relationship to recurrence and its clinical value in predicting prognosis. The tests of immune competence used were immunoglobulins IgG, IgA, IgM, leucocyte counts, percentage and total lymphocyte counts and Mantoux and DNCB skin hypersensitivity tests. None of these tests was strongly predictive of recurrence on an individual basis, a finding similar to our results at 2 years. The longer period of follow-up now reported has provided no findings of unequivocal statistical significance, but suggests a biphasic host response to early tumours. The patients who developed recurrence within 5-11 years due to micrometastasis had higher lymphocyte counts in their preoperative assessment than patients who remained recurrence free. This suggests that small tumour volumes do not stimulate immunity and that large volumes depress it; tumours in between these groups are associated with higher levels. Examination of studies by a number of authors reveal parallel findings which have not been previously noted. It is not possible to confirm the significance of these findings from this study because of the heterogeneity of human breast cancer. However, if they indicate a general principle of a dynamic host-tumour interplay they have important implications for assessing immune competence at any single point of time and for the theory that cancer may arise during an anergic state. We hope that these findings will stimulate other workers to examine host-tumour interaction from this point of view.

Breast Neoplasms↗
PubMed1986

Long-term follow-up of general immune competence in breast cancer. II. Sequential pre- and post-treatment levels: a 10 year study.

Pre-treatment and sequential post-treatment (at 3 months, 6 months, 1, 2, 3, 4 and 5 years) examination of general immune competence was performed in 185 consecutive breast cancer patients. The patients were followed for 5 to 11 years to monitor the dynamic relationship between host immunity and cancer and to examine the effect of the treatment method. The tests of immune competence used were immunoglobulins IgG, IgA, IgM, leucocyte counts, percentage and total lymphocyte counts and Mantoux and DNCB skin hypersensitivity tests. Serum IgG and IgA showed no change relating to treatment method in recurrence-free patients; but IgG levels were higher when recurrent disease was imminent or established; IgM diminished (P less than 0.001) after treatment and this continued at 5 years in all patient groups. Simple lymphocyte counts showed the most interesting changes. They remained depressed for as long as 60 months following radiotherapy (P less than 0.01). After treatment by surgery, lymphocyte counts rose in patients without recurrence, but fell when systemic recurrence was imminent or established. This effect was not seen in patients with local recurrence only. There was no change in immune competence immediately before recurrence sufficient to be of clinical usefulness, but a low pre-treatment lymphocyte count with a steady rise after surgery carried a good prognosis. Similarly a high initial lymphocyte count with a fall after surgery was indicative of recurrence. Universal and prolonged depression of lymphocyte counts following radiotherapy was confirmed, and the effect was additive to that of tumour load in recurrent disease. Because of the large number of statistical calculations carried out, some of the apparently significant findings may be due to chance. However, the general trends emerging suggest that similar long-term studies, using the more sophisticated measures of lymphocyte function now available, might be rewarding.

Breast Neoplasms↗
PubMed1986

Effects of short chain fatty acids on a new human colon carcinoma cell line (LIM1215).

The effects of short chain fatty acids on a colon carcinoma cell line, LIM1215, have been studied. Of the four short chain fatty acids tested only butyrate at 1 mmol/l and 10 mmol/l and acetate at 10 mmol/l had significant effects on this cell line. The addition of butyrate to growth medium affected the growth rate and the production of alkaline phosphatase, dipeptidyl peptidase IV and carcinoembryonic antigen. Butyrate at a final concentration of 1 mmol/l increased the doubling time of the cells from 26 hours to 72 hours and decreased the cloning efficiency of the cells from 1.1% to 0.054%. Alkaline phosphatase concentrations increased rapidly in cells cultured in 1 mmol/l butyrate reaching peak levels after four days with alkaline phosphatase concentrations increasing more than six-fold. Levels of dipeptidyl peptidase IV and carcinoembryonic antigen were also increased after culture in butyrate containing medium. The number of alkaline phosphatase containing and dipeptidyl peptidase IV containing cells increased markedly in butyrate containing cultures. In contrast the number of mucus containing cells decreased in cultures grown in medium containing butyrate. This differentiating effect of butyrate on colon carcinoma cells may be relevant to the presence of butyrate in the colonic contents and the relationship between short chain fatty acids and fibre intake.

Alkaline Phosphatase↗