PubMed Health⌕ Search

Biomedical subjects

R M Hoffman

Publications and source records attributed to R M Hoffman.

At least 145 records · Page 8Linked to original sources
PubMed1992

Characterization of the human pleiotrophin gene. Promoter region and chromosomal localization.

The protein (PTN) encoded by the pleiotrophin (PTN) gene belongs to a recently described family of heparin-binding cytokines whose expression is temporally and spatially regulated during development. We have now isolated genomic clones of the human PTN gene, characterized its promoter region, determined its transcription initiation site(s), and established functional activity of the PTN promoter. A fragment -550/+191 that contains a CAAT box, no apparent TATA box, and four consensus sites for the binding of MyoD is sufficient to provide optimal promoter activity. A serum response element is found at -559 to -568. We also have identified the human PTN gene on chromosome 7, band q33 and the mouse Ptn gene on chromosome 6, respectively. The data thus identify and characterize the 5' end of the PTN gene and its promoter region, suggest potential regions that may contribute to the regulation of its transcriptional activity, and localize the PTN gene in human and mouse chromosomes.

3T3 Cells↗
PubMed1992

Hair shaft elongation, follicle growth, and spontaneous regression in long-term, gelatin sponge-supported histoculture of human scalp skin.

In order to better understand the molecular mechanisms of human hair growth control and to test hair growth-modulatory drugs, appropriate in vitro models are required. Here, we report the long-term growth, shaft elongation, and spontaneous regression of human hair follicles in histoculture of intact scalp skin. Human scalp skin with abundant hair follicles in various stages of the hair growth cycle was grown for up to 40 days in a gelatin sponge-supported histoculture system at the air/liquid interface. Isolated follicles placed in the gelatin-sponge matrix also supported hair shaft elongation, with the hair follicle cells remaining proliferative and viable for very long periods. Hair shaft elongation occurred mainly during the first 10 days of histoculture of both intact skin and isolated follicles. However, hair follicles were viable and follicle keratinocytes continued to incorporate [3H]thymidine for up to several weeks after shaft elongation had ceased as shown by fluorescent-dye double staining, measured by confocal laser scanning microscopy, and by histological autoradiography of [3H]thymidine incorporation, respectively. Hair follicles could continue their cycle in histoculture; for example, apparent spontaneous catagen induction was observed both histologically and by the actual regression of the hair follicle. In addition, vellus follicles were shown to be viable at day 40 after initiation of culture. In the histocultured human scalp we demonstrated the association of mast cells with anagen follicles and macrophages with catagen follicles, suggesting a role of these cells in the hair cycle. This histoculture technique should serve as a powerful tool for future hair research in the human system as well as a screening assay for compounds that can perturb the hair cycle.

Culture Techniques↗
PubMed1992

Noncolorimetric measurement of cell activity in three-dimensional histoculture using the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide: the pixel image analysis of formazan crystals.

We describe a novel system for measuring the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction in three-dimensional histoculture which is no longer dependent on colorimetric determination of extracted formazan, but rather is based on a pixel image analysis of formazan crystals, and which allows intratumor heterogeneity to be taken into account. The MTT test is based on the enzymatic reduction of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheniltetrazolium bromide to formazan crystals by living, metabolically active cells, but not in dead cells. The reaction was carried out in situ in six-well plates on gel-supported histocultured human tumors. After a 24-h incubation with different drugs the tumors were incubated with a solution of MTT. Frozen sections of the tumor pieces were made and the slides were then stained with a propidium iodide solution, whose fluorescence is proportional to the number of cells present. We demonstrate here that the formazan crystals, formed by MTT reduction, reflect polarized light and that this can be quantified by using an image analysis system based on bright-pixel quantitation directly on a frozen section of the original tissue. Combined with the use of the fluorescent dye propidium iodide, also measured by pixel analysis, we can express a ratio between the total amount of MTT reduction and the total number of cells present in the specimen that expresses the effect of drugs on the histocultured tumors. Since histology is well maintained in histoculture it is possible to take into account the heterogeneity present in the tumor with regard to drug response.(ABSTRACT TRUNCATED AT 250 WORDS)

Antineoplastic Agents↗
PubMed1992

A metastatic nude-mouse model of human pancreatic cancer constructed orthotopically with histologically intact patient specimens.

Pancreatic cancer is one of the most intractable and least understood of all human cancers. Pancreatic cancers is the fourth-leading cause of cancer-related mortality in the United States with less than 2% of the patients surviving for 5 yr. In an effort to help develop more effective treatment modalities for pancreatic cancer and improve detection, we report an animal model for individual human pancreatic-cancer patients. The model involves orthotopic transplantation of histologically intact pancreatic-cancer specimens to the nude-mouse pancreas, which can result in models that resemble the clinical picture including (i) extensive local tumor growth, (ii) extension of the locally growing human pancreatic cancer to the nude-mouse stomach and duodenum, (iii) metastases of the human pancreatic tumor to the nude-mouse liver and regional lymph nodes, and (iv) distant metastases of the human pancreatic tumor to the nude-mouse adrenal gland, diaphragm, and mediastinal lymph nodes. In a series of five patient cases, a 100% take rate has been demonstrated, and of 17 mice transplanted, 15 supported tumor growth. Immunohistochemical analysis of the antigenic phenotype of the transplanted human pancreatic tumors showed a similar pattern of expression of two different human tumor-associated antigens, such as tumor-associated glycoprotein 72 and carcinoembryonic antigen in the transplanted tumors when compared with the original surgical biopsy, suggesting similarity between the two. This model should, therefore, prove valuable for treatment evaluation of individual cancer patients, as well as for evaluation of experimental treatment modalities for this disease.

Adenocarcinoma↗
PubMed1992

The v-sis oncogene product but not platelet-derived growth factor (PDGF) A homodimers activate PDGF alpha and beta receptors intracellularly and initiate cellular transformation.

The v-sis oncogene product p28v-sis and the platelet-derived growth factor (PDGF) B chain share 92% homology with each other and over 50% homology with the PDGF A chain. Exogenously added homodimers of PDGF A and PDGF B and of p28v-sis are potent mitogens but only PDGF B and p28v-sis induce transformation when endogenously expressed with a strong promoter. Because exogenous PDGF AA and PDGF BB both initiate a full mitogenic response, understanding the mechanisms underlying the difference in their transforming potential may clarify how growth factor genes act as oncogenes. In this work, we compared cells expressing high levels of PDGF A and v-sis. We observed that transformation by v-sis correlated directly with the rapid degradation (t1/2 approximately 20 min) of the alpha and beta PDGF receptors, with a failure of either the alpha or beta receptor to be fully processed and with the association of high levels of phosphatidylinositol (PI) 3-kinase with immunoprecipitates of the PDGF receptors. In contrast, in cells expressing essentially equal levels of PDGF A, transformation was not detected, alpha and beta PDGF receptor processing was normal, and association of PI 3-kinase with receptors in immunoprecipitates was not found above control values. The ability of v-sis to autoactivate PDGF receptors within processing compartments and to initiate activation of the PI 3-kinase signaling pathway coupled with the failure of PDGF A to activate its receptor intracellularly and to induce transformation when endogenously expressed at high levels suggests that the internal autoactivation of PDGF receptors may be essential for transformation by v-sis.

3T3 Cells↗
PubMed1992

Measurement of androgen sensitivity in the human prostate in in vitro three-dimensional histoculture.

We have adopted an in vitro three-dimensional histoculture technique for assay of androgen sensitivity in explants of human benign prostatic tissue. The assay is based on the uptake of 3H-thymidine/micrograms protein in explants of prostate incubated in parallel with dihydrotestosterone (DHT) and hydroxyflutamide (HF) controls. The ratio of 3H-thymidine/micrograms protein in DHT treated samples per 3H-thymidine/micrograms protein in HF treated samples provides an index of androgen sensitivity. The DHT/HF index measured in 24 BPH specimens averaged 3.6. To determine the specificity of the HF effect, we measured the DHT/HF index in a single prostate at different concentrations of HF in the presence of fixed concentrations of DHT (2 x 10(-8) M) and noted a dose-response relationship. In addition we noted no effects of HF on 3H-thymidine incorporation over a range of 2 x 10(-4)M compared to 2 x 10(-7)M, except at the highest concentration. Of surprise was the finding of an average DHT/HF index in 5 different nonprostate tissues, including breast, uterus, colon, kidney, and thyroid, that was similar to the index found in prostates. We plan to adapt this androgen sensitivity assay to measure the DHT/HF index in biopsy-size samples of prostate, since such an assay could then be utilized to determine androgen sensitivity in individual patients with prostate cancer.

Androgen Antagonists↗
PubMed1991

Models of human metastatic colon cancer in nude mice orthotopically constructed by using histologically intact patient specimens.

There is an important need for clinically relevant animal models for human cancers. Toward this goal, histologically intact human colon-cancer specimens derived surgically from patients were implanted orthotopically to the colon or cecum of nude mice. We have observed extensive orthotopic growth in 13 of 20 cases of implanted patient colon tumors. These showed various growth patterns with subsequent regional, lymph-node, and liver metastasis, as well as general abdominal carcinomatosis. Thus, models for human colon cancer have been developed that show (i) local growth, (ii) abdominal metastasis, (iii) general abdominal carcinomatosis with extensive peritoneal seeding, (iv) lymph-node metastasis, (v) liver metastasis, and (vi) colonic obstruction. These models permit the passage of the tumors to form large cohorts. They will facilitate research into the biology of colon cancer metastatic capability and the development of new drugs active against metastatic cancer. These models may also predict the clinical course and the in vivo response to drugs of the cancer of individual patients.

Abdominal Neoplasms↗
PubMed1991

Correlation of histology and drug response of human tumors grown in native-state three-dimensional histoculture and in nude mice.

An in vitro histoculture system in which a native-state collagen-sponge gel supports the three-dimensional growth of tumor tissue has been recently developed that allows the culture and drug response assay for most every tumor type. Important features of the histoculture system include the maintenance of three-dimensional tissue architecture and the use of histological autoradiography to measure nuclear incorporation of [3H]thymidine as an endpoint. We describe in this report in vitro-in vivo correlations for drug response and tumor histology by using human tumor xenografts grown in the native-state three-dimensional histoculture system and as xenografts in nude mice. This comparison eliminates many of the confounding variables seen in most correlative clinical trials. Results demonstrate (i) a very high preservation of in vivo tissue architecture in vitro, (ii) an 86% accuracy in vitro of predicting drug resistance in vivo using suprapharmacologic doses of drugs in vitro, and (iii) an overall predictive frequency of drug resistance and sensitivity ranging from 53% for 5-fluorouracil to 78% for doxorubicin.

Animals↗
PubMed1991

Skin toxicity determined in vitro by three-dimensional, native-state histoculture.

We describe a gel-supported in vitro system for culturing skin samples in a three-dimensional native state. All the cell types of skin remain viable and maintain their native architecture for at least 10 days. The culture system is used for toxicity measurements by ascertaining cell viability using two fluorescent dyes: 2',7'-bis-(2-carboxyethyl)-5-(and -6)carboxyfluorescein acetoxymethyl ester, specific for living cells, and propidium iodide, specific for dead cells. Cell staining with the dyes is measured throughout the tissue block by confocal scanning fluorescence microscopy. The dose-response to three agents--ethanol, doxorubicin, and sodium hypochlorite--is shown and, in the case of sodium hypochlorite, compared to in vivo skin toxicity with a high correlation. We also demonstrate that the end point of [3H]thymidine incorporation measured by histological autoradiography can be used to measure toxicity. Our results with the [3H]thymidine end point demonstrate that the hair follicle cells are the most sensitive to doxorubicin. The native-state model for skin may be an effective replacement for animal systems and superior to the dispersed skin cell systems used previously. It can allow rapid, inexpensive measurements of the effect of manufactured products, drugs, and pollutants on skin.

Animals↗
PubMed1991

Drug response of head and neck tumors in native-state histoculture.

We describe a chemosensitivity testing of head and neck tumors, in which a native-state histoculture, ie, a three-dimensional culture system that maintains important in vivo properties, including tissue architecture, was used. Fifteen specimens of head and neck tumors were evaluated for sensitivity to the following drugs: cisplatin (DDP) at concentrations of 1.5, 15, and 37.5 micrograms/mL; fluorouracil at concentrations of 4.0, 40, and 100 micrograms/mL; and combinations of cisplatin and fluorouracil in corresponding doses. Growth and measurement of drug responses were successfully completed in 10 specimens (five others were contaminated, four of them prior to instituting rigorous antibiotic washes). The results indicated cisplatin sensitivity in five of 10 patients; fluorouracil sensitivity in four of 10 patients; and fluorouracil-cisplatin sensitivity in seven of eight patients. Our preliminary results indicate that the native-state histoculture technique is feasible to test chemosensitivity of head and neck tumors.

Antineoplastic Combined Chemotherapy Protocols↗