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R H Whitehead

Publications and source records attributed to R H Whitehead.

At least 19 recordsLinked to original sources
PubMed1992

Cell-surface fucosylation and magnetic resonance spectroscopy characterization of human malignant colorectal cells.

Proton (1H) magnetic resonance spectroscopy (MRS) has been used to distinguish lowly and highly tumorigenic human malignant colorectal cell lines based on differences in lipid, choline, and fucose resonances. The spectral patterns were comparable with those obtained for human colorectal biopsy specimens, indicating that cells grown in vitro are suitable for documenting colorectal tumor biology. For the first time, two-dimensional (2D) correlation spectroscopy (COSY) has been used to assess the fucosylation state on the surface of intact viable cells, and differences were recorded between the highly and lowly tumorigenic cell lines. Four methyl-methine cross-peaks were assigned to covalently linked fucose on the basis of increases in volume following the addition of free fucose. Both cell lines incorporated the same amount of exogenous free fucose as determined chemically, but the COSY spectra indicated that the fucose was distributed differently by each cell line. Of the four sites containing MR-visible bound fucose, one was common to both cell lines, two characteristic of the highly tumorigenic line, and the remaining site unique to the lowly tumorigenic cells. Material released from the highly tumorigenic cells in response to increased cell density was also fucosylated (whereas shed material from lowly tumorigenic cells was not), suggesting a biological role for shed fucosylated antigens in tumor aggression.

Amino Acids↗
PubMed1992

Patterns of growth and differentiation in the colon carcinoma cell line LIM 1863.

The LIM 1863 colon carcinoma cell line grows in the form of morphologically and functionally organized organoids. Cells are arranged around a central lumen with a brush border and nuclei are polarized to the periphery. The organoids contain 3 morphological cell types (columnar, goblet and caveolated cells). By agar cloning it has been possible to isolate 29 subclones of the cell line, all of which display the same phenotype and percentage of morphological cell types as the parent line. Cell-sorting experiments showed that precursor cells of LIM 1863 cultures could express either mucin (large-intestinal-mucus antigen) or a brush-border enzyme (sucrase-isomaltase). Proliferating cells were predominantly found near the outer periphery of organoids with cell maturation towards the internal lumen. Dead cells were continuously shed from the organoids but terminal non-cycling cells were not observed within the organoids. The organoid structure was calcium-dependent and promoted cell survival. Suspension cultures of disaggregated cells could be grown in medium containing less than 100 microM calcium. No decrease in differentiation antigens was observed in the low-calcium cultures, although polarization of the cells was lost. The organoid cultures formed by this cell line represent a unique in vitro model for colonic crypt growth and organization.

Calcium↗
PubMed1992

SMS 201.995 inhibits in vitro and in vivo growth of human colon cancer.

The effect of a long-acting somatostatin analogue SMS 201.995 (SMS; Sandoz) on basal and gastrin-stimulated growth of 4 human colon cancer lines was studied in vitro and in vivo. Proliferation assay was done with overnight [75Se]selenomethionine uptake after 5 days of incubation. Gastrin concentrations used were 5e-10 M and 1e-7 M. SMS concentrations were from 2e-12 M to 2e-7 M. Cell lines LIM 1215, LIM 2405, and LIM 2412 were inhibited dose-dependently in both basal and gastrin-stimulated groups. LIM 1863 was slightly stimulated. Based on in vivo growth characteristics, LIM 2412 and LIM 2405 were selected for xenograft study. The dose of 50 micrograms/kg/day was arrived at after a preliminary experiment showed it to be safe and effective. The LIM 2412 xenografts in the SMS-treated animals were 473.3 +/- 99.9 (SD) versus 838.1 +/- 111.3 mm3 in control (P less than 0.05) after 20 days. The LIM 2405 tumors were also significantly inhibited (81.2 +/- 30.0 versus 245.7 +/- 48.3 mm3, P less than 0.01). The effect of SMS appeared to be reversible. Oral SMS at 200 micrograms/kg/day was not absorbed. This study suggests that SMS may have direct antitumor effects in human colon cancer.

Animals↗
PubMed1992

Transplantation of fetal mouse colon under the kidney capsule of an adult mouse: a model for the study of colonic development.

Fourteen-day fetal mouse colon was transplanted under the kidney capsule of an adult mouse to determine whether this system could be used as a model of embryonic colonic development. The 14-day fetal colon was transplanted and left for a period of 7 days. Comparisons of the normal one day postnatal colon and the transplanted colon were made morphologically and morphometrically. It was found that the transplanted fetal colon resembled its postnatal counterpart with respect to morphology; the cell types seen in the transplanted colon were similar to those observed in the in situ colon of the same age. However, morphometric analysis showed that the transplanted colon was significantly smaller than its postnatal counterpart, suggesting that conditions in the host were not optimal to support the full growth of the colon. In spite of this, it appears that the fetal colon can differentiate normally under the kidney capsule and this model can be used to study both epithelial-mesenchymal interactions and the role of hormones in fetal colonic development.

Animals↗
PubMed1992

Retention of tissue-specific phenotype in a panel of colon carcinoma cell lines: relationship to clinical correlates.

A panel of eight cell lines has been derived from colon carcinomas. These cell lines have both been characterized according to standard criteria of growth rate, response to mitogens (epidermal growth factor and basic fibroblast growth factor), xenograft growth and growth in soft agar, and according to the ability of the cells to express epitopes known to be expressed by cells in the normal intestinal mucosa. The expression of epitopes present in columnar (absorptive) cells has been assessed using a panel of monoclonal antibodies to brush border peptidases and disaccharidases, villin and brush border-specific peptides. Goblet cell epitopes have been determined by monoclonal antibodies to mucin and carcinoembryonic antigen. An antibody to chromogranin was used to identify endocrine cells. Using these antibodies we found that all the cell lines reacted with at least one of the antibodies to columnar cells. Similarly, varying proportions of cells in six of the eight cell lines stained with antibodies to mucin. None of the cells expressed chromogranin. Expression of a differentiated colonic phenotype, as measured from antibody staining, did not correlate with measurements of malignancy, such as the ability of the cells to grow in soft agar or as xenografts. Similarly, there was no correlation between retention of a colonic phenotype and the initial pathological stage of the tumour from which the cell lines were derived.

Animals↗
PubMed1992

Expression of MUC1 and MUC2 mucins by human tumor cell lines.

The secretion and nature of mucins produced from a panel of recently available new gastric and colon carcinoma cell lines (LIM1839, LIM1215, LIM1863, LIM1899, LIM2099, LIM2405, LIM2408, LIM2412, LIM2463), as well as other colon (LS174T, HT29, HT29-SB, COLO533, COLO206), breast (T47D, MCF-7, BT20, ZR75-1) and ovarian (COLO316) tumor cell lines, was investigated. ELISA and Western blotting of the culture supernatants with novel anti-MUC1 and anti-MUC2 monoclonal antibodies (MAbs) specific for mucin core proteins showed their secretion by most of these cell lines. In addition, mucins produced by these cell lines expressed the tumor-associated carbohydrate detected by MAb 3E1.2 (glycolylsialyl-Tn, mammary serum antigen or MSA) and the Tn or T antigens reactive with lectin SSA-M. SSA-M detected MUC1 or MUC2 captured by MAbs BC2 or CCP58, while 3E1.2 only detected MUC1-associated carbohydrate, indicating that the MAb may react with a conformationally dependent epitope, or that the sialyl/glycolyl-transferases involved in MSA production may be sequence specific. In addition, the BC2/SSA-M and CCP58/SSA-M assays detected mucins in some samples which were not detected by BC2/BC2 or CCP58/CCP58 dual determinant assays, indicating that this format may be more appropriate for the detection of tumor-associated mucins in body fluids. These new cell lines and assays should be of use in the investigation of mucin core proteins, particularly LIM2463 and LIM1839 which express significant quantities of both MUC1 and MUC2.

Amino Acid Sequence↗
PubMed1992

Effects of cholera toxin on human colon carcinoma cell lines.

This study reports on changes in morphology and membrane transport in 5 human colon carcinoma cell lines treated with cholera toxin (CT). Three of the cell lines that grew as monolayers (LIM 1215, LIM 1899, LIM 2099) and 1 that grew as floating clumps (LIM 2408) did not show morphological changes after CT treatment. However, cell line LIM 1863 that grows as floating "crypt-like" organoids showed rapid and distinctive changes in morphology and membrane transport after CT treatment. At 1 and 6 hrs after CT treatment, light and transmission electron microscopy revealed rapid dilatation of the central lumen of organoids and the appearance of 2 populations of apical vesicular inclusions. The first population was unusual in being non-membrane bound and limited by fuzzy filamentous material. The second population was membrane bound. Scanning electron microscopy at 1-6 hr after CT treatment showed swelling and loss of surface microvilli on some, but not all, cells. At 24 hr after CT treatment the majority of organoids showed evidence of fluid accumulation and small apical vesicles coalesced to form large single vacuoles that obliterated normal cell morphology. By 48 hr, continued swelling produced extreme attenuation of the plasma membrane with cells taking on an "endothelial cell-like" appearance. The response to CT was dose-dependent. Uptake studies using 86Rubidium and blocking studies using ouabain and amiloride indicated that CT is acting on the Na+/K+ ATPase membrane pump to cause the increased fluid uptake by LIM 1863 cells. This study is the first to report specific morphological changes in intestine-derived cells in response to CT.(ABSTRACT TRUNCATED AT 250 WORDS)

Carcinoma↗
PubMed1992

Differential regulation of transforming growth factor alpha autoinduction in a nontransformed and transformed epithelial cell.

Addition of transforming growth factor alpha (TGF-alpha) to cultured human keratinocytes results in enhanced expression of TGF-alpha mRNA. This phenomenon of TGF-alpha autoinduction is also observed in a TGF-alpha responsive colon cancer cell line, LIM 1215. In the present study, regulation of TGF-alpha autoinduction is examined in these two cell types. In human keratinocytes, but not in LIM 1215 cells, the increase in steady-state TGF-alpha mRNA following administration of TGF-alpha is due to stabilization of the 4.8-kilobase TGF-alpha transcript, as determined by actinomycin D decay curves. Nuclear run-on experiments confirmed transcriptional control in LIM 1215 cells. Basal and TGF-alpha-stimulated TGF-alpha expression is mediated, at least in part, through a protein kinase C-dependent pathway in both cell types, as determined by the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), which attenuates TGF-alpha mRNA accumulation. In the keratinocytes, but not in the LIM 1215 cells, basal TGF-alpha expression is mediated through an epidermal growth factor receptor-dependent pathway, as determined by antibody blockade of the epidermal growth factor receptor. Thus, differential regulation of TGF-alpha autoinduction exists in these nontransformed and transformed epithelial cell types.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Myb expression is higher in malignant human colonic carcinoma and premalignant adenomatous polyps than in normal mucosa.

Expression of the protooncogene c-Myb protein was assessed in normal mucosa and in tumor samples resected from six patients. We found that the tumor samples always expressed higher levels of full length Myb protein than the normal tissue. This contrasts with the situation in c-myb-associated hemopoietic malignancies of the mouse and chicken, in which Myb proteins are generally amino or carboxyl truncated. Tissues from five patients with colonic adenomatous polyps were also examined and found to express levels of Myb that were, in general, intermediate between those found in normal tissues and tumors. Of particular interest is that the more dysplastic polyps displayed higher Myb levels. In one patient with carcinoma and multiple colonic polyps, some polyps had intermediate levels of Myb, whereas one polyp with carcinoma in situ expressed tumor-like levels of Myb. To directly test the hypothesis that Myb expression may be important in determining the rate of colonic cell proliferation, we examined three colonic carcinoma cell lines and one polyp cell line. We found that the cell lines with the most rapid doubling times exhibited the highest Myb levels. In addition, we show that antisense myb oligonucleotides retard the proliferation of one of these colonic cell lines which expresses the highest level of Myb.

Base Sequence↗
PubMed1991

The major colonic cell mitogen extractable from colonic mucosa is an N terminally extended form of basic fibroblast growth factor.

Colonic growth factors (CGFs) were extracted from porcine intestinal epithelium and mucosa. Under acidic conditions, very little mitogenic activity (as assayed using murine 3T3 fibroblasts and a human colonic cell line) was extractable. However, by extracting at neutral or slightly alkaline pH, significant mitogenic activity for both the murine fibroblasts and human colonic carcinoma cell line could be detected. CGFs are present throughout the intestine and cecum. The epithelial mucosa of the distal colorectal region appeared to contain mitogens which were more potent for the colonic cells than the 3T3 fibroblasts. Purification of CGFs from the colonic mucosa required removal of associated mucin by pH precipitation prior to chromatographic fractionation. It was then possible to develop a complete purification (390,000-fold) scheme for the major CGF, an 18-kDa protein which bound to heparin-Sepharose. N-terminal sequence analysis yielded a single sequence (Q)SPGGAMAAGSITTLPALP, i.e. an N-terminally extended form of basic fibroblast growth factor. Apart from the substitution of Gly in bovine basic fibroblast growth factor by a Ser in porcine CGF, the proteins are identical. A similar extraction procedure using purified human colonic crypt epithelial cells yielded a mitogen for the human colonic cell line with similar chromatographic properties.

Amino Acid Sequence↗
PubMed1991

A cell line (LIM 2463) derived from a tubulovillous adenoma of the rectum.

We describe a new epithelial cell line (LIM 2463) derived from tubulovillous adenoma of the rectum. The cells grow as organoids and secrete large amounts of mucus. The cells are polarized, with a microvillar brush border, and express dipeptidyl peptidase IV in a polarized manner. No staining was seen with 3 other antibodies directed against other brush-border hydrolases or disaccharidases. Focal polarized staining was obtained with 2 antibodies directed against other brush-border-associated peptides. The cells are aneuploid with a distinctive karyotype (48, XX, +9, +13). All attempts to clone the cells in semi-solid agar or to grow them as xenografts in nude athymic mice have failed.

Adenoma↗
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↗
PubMed1979

Significance of tumour mass on T-lymphocyte levels in patients with gastrointestinal cancer.

The relationship between tumour load and immunity in gastrointestinal cancer has been studied by sequential comparison in patients whose tumour has been removed and those whose tumour was found to be inoperable. Total lymphocyte count, absolute and percentage T- and B-lymphocyte counts, effect of papain on E-rosetting cell levels, and inhibitory effect of cancer sera on E-rosette formation by normal lymphocytes have been studied in 30 patients with stomach or colorectal cancer, and 10 control patients with benign gastrointestinal disease. The examination was done on each patient before and at regular intervals after operation up to 24 weeks. Operable cases, with removal of tumour load, showed a temporary fall in total lymphocyte count and T cell counts, which returned to normal by four weeks postoperatively. Inoperable cases (15 patients) showed a progressive fall in total lymphocyte count and a relatively greater depression of T cell counts, in parallel with increasing tumour mass. E-receptor blocking factor was demonstrated in the sera of cancer patients. This factor was related to tumour mass and presumably was of tumour origin, as it persisted in the inoperable group but disappeared by 12 weeks after tumour removal. The factor explained the excess depresion of T cells over total lymphocytes, but does not explain the continuing depression of total lymphocyte count in the cancer patients.

Aged↗
PubMed1978

Importance of methodology in demonstrating depression of T-lymphocyte levels.

A comparison has been made of 3 methods of determining E rosettes in young, healthy people, women with breast cancer and an age-matched group of healthy women, in an attempt to explain the wide variations in T-cell levels in different disease states reported by different workers. The greatest difference in levels of E-rosetting cells between the different groups was seen in incubation of 1 1/2 h at 4 degrees C. Much of the difference seen in these comparisons disappeared after overnight incubation at 4 degrees C, which was associated with an increased T-cell level in all groups. Consequently, although maximal levels of T lymphocytes as determined by E rosetting are found using overnight incubation, a short incubation period may be superior for demonstrating subtle depressions in levels of T lymphocytes as seen in elderly people and cancer patients. This depression is not considered to be cancer specific, because of the findings in the age-matched control group and similar findings in benign disease states.

Adult↗
PubMed1978

The macrophage electrophoretic mobility (MEM) test--an investigation of its value as a routine laboratory test in the detection of malignant disease.

A previous study of the MOD-MEM test showed promising results. We have attempted to repeat the study using a blind coded series of 210 blood samples from normal subjects and patients with either benign or malignant disease. Using standard criteria the false negative rate for cancer patients averaged 43% and the false positive rate for non-cancer patients averaged 34%. The results indicate that the test at the present time, under routine laboratory conditions, is not reliably reproducible and does not have the ability to effectively discriminate between benign and malignant disease. It is suggested that blind coded studies be used more frequently in assessment of tests with cancer detection potential.

Antigens↗
PubMed1977

Distribution of 5-fluorouracil to body tissues compared after intraluminal, intravenous, and intramural administration in gastrointestinal cancer.

The distribution of 5-fluorouracil (5-FU) to various potential metastatic sites has been investigated in stomach and colorectal cancer patients, using intraluminal, intravenous, and intramural routes of administration at the time of surgery. The best overall concentrations of 5-FU in systemic blood, portal blood, liver, draining lymph nodes, tumor, and locally in the bowel wall was obtained after intramural injection. The intraluminal route of administration resulted in poor overall distribution of 5-FU. Intravenous administration tended to give levels between those obtained from intramural and intraluminal routes. There was no complication of 5-FU therapy. On the basis of this study we conclude that the intramural route is the most satisfactory for adjuvant administration of 5-FU with surgery. This might be expected to give better results than those obtained in clinical studies using intraluminal and intravenous routes. A controlled trial of 5-FU using the intramural route is indicated in gastric and colorectal cancer to demonstrate whether or not clinical benefit can be obtained.

Aged↗