Defective ubiquitinylation of EGFR mutants of lung cancer confers prolonged signaling.

Several distinct mutations within the kinase domain of the epidermal growth factor receptor (EGFR) are associated with non-small cell lung cancer, but mechanisms underlying their oncogenic potential are incompletely understood. Although normally ligand-induced kinase activation targets EGFR to Cbl-mediated receptor ubiquitinylation and subsequent degradation in lysosomes, we report that certain EGFR mutants escape this regulation. Defective endocytosis characterizes a deletion mutant of EGFR, as well as a point mutant (L858R-EGFR), whose association with c-Cbl and ubiquitinylation are impaired. Our data raise the possibility that refractoriness of L858R-EGFR to downregulation is due to enhanced heterodimerization with the oncogene product HER2, which leads to persistent stimulation.

Nuclear and cytoplasmic expression of ErbB-4 in prostate cancer.

PURPOSE: To evaluate cytoplasmic and nuclear ErbB-4 expression in prostate cancer specimens and its association with outcome. BASIC PROCEDURES: Specimens of 50 prostate cancer patients were investigated for ErbB-4 overexpression using Immunohistochemistry staining. Cytoplasmic and nuclear staining was graded as 0-3 according to its intensity. The prognostic parameters were tumor stage, PSA level, Gleason score, probability of positive lymph nodes (Partin's tables and Roach equation), and 5-year disease free survival (Kattan nomogram). MAIN FINDINGS: Overexpression of ErbB-4 (> or = 1) was detected in 30 (60%) patients and overexpression using cytoplasmic and nuclear staining was > or = 2 in 19 (38%) and 17 (34%) patients, respectively. In only one third of the specimens was there any similarity between the 2 types of staining. Advanced tumor stage, high pretreatment PSA levels and high Gleason scores were evenly distributed among the patients with low (< or = 1) and intermediate/high (> or = 2) ErbB-4 expression. The probability of lymph node involvement and 5-year disease free survival were similar in both types of staining. PRINCIPAL CONCLUSIONS: ErbB-4 was overexpressed (cytoplasmic and nuclear staining) in approximately one third of prostate cancer patients. The rate of similarity between the 2 staining types was only 33%: overexpression was evenly distributed among intermediate/high and low risk prostate cancer patients with both staining methods.

Evaluation of skin viscoelasticity and anisotropy by measurement of speed of shear wave propagation with viscoelasticity skin analyzer.

Skin viscoelasticity was evaluated by a fast, noninvasive assay based on the measurement of the speed of elastic shear wave propagation in the skin by a new portable and user-friendly viscoelasticity skin analyzer. The range of speed of elastic shear wave propagation measured by viscoelasticity skin analyzer allows the evaluation of the stiffness of a wide spectrum of artificial materials as well as the viscoelasticity of skin of laboratory animals and human subjects. The directional nature of the measurement enables to monitor the anisotropy of the materials tested. The speed of elastic shear wave propagation was shown to have a positive correlation with the stiffness of the material tested. In symmetric contralateral areas of intact skin in rabbit ears, similar viscoelasticity and anisotropy were observed. Twenty-four hours after the induction of local edema by croton oil, skin stiffness and anisotropy were significantly increased. In healthy human subjects of both sexes significant variations in skin stiffness and anisotropy were observed in three different skin areas along the forearms, but the speed of elastic shear wave propagation was similar in the symmetric contralateral areas. Age (17-65 y) seemed to have a limited effect on the viscoelasticity of the forearm skin. Hydrating creams decreased the stiffness of the forearm skin for only approximately 3 h. The stiffness and anisotropy of the skin of the breasts in female volunteers (20-86 y) increased with age, but the speed of elastic shear wave propagation was similar in symmetric contralateral areas in the same individuals. Based on these results, we propose the application of the viscoelasticity skin analyzer in experimental and clinical practice for quantitative evaluation of skin condition.

Fibrin microbeads (FMB) as biodegradable carriers for culturing cells and for accelerating wound healing.

We have developed biodegradable fibrin-derived microbeads as potent cell carriers. The fibrin-derived microbeads, 50-200 microm in diameter, were tested for their attachment to a wide range of cell types. Fibrin-derived microbeads were shown to be greatly haptotactic to cells (such as endothelial cells, smooth muscle cells and fibroblasts), which respond to fibrinogen in contrast to keratinocytes and different cell lines derived from leukocytic lineage. The cells on fibrin-derived microbeads could be maintained for more than 10 d and achieved a high density. 31P-nuclear magnetic resonance was employed to monitor phosphate metabolism in cells, with densities on the order of 100 million cells per g of fibrin-derived microbeads. The 31P-nuclear magnetic resonance adenosine triphosphate and phosphocreatine signals, equivalent to the signal obtained with perfused normal skin, indicated that metabolism of cells on fibrin-derived microbeads was responsive to oxygenation and nutrients. Light, fluorescent, and confocal laser microscopy revealed that the porous fibrin-derived microbeads accommodate up to 200-300 cells due to their high surface area which minimized contact inhibition. Cells could degrade the fibrin-derived microbeads and be transferred to seed culture flasks without trypsinization. In a pig skin wound healing model, fibrin-derived microbeads + fibroblasts were transplanted into full thickness punch wounds. This procedure was compared with other treatment modalities, such as the addition of human platelet-derived growth factor BB or fibrin-derived microbeads alone. By the third day after wounding, only the wounds in which fibroblasts on fibrin-derived microbeads were added showed significant formation of granulation tissue. Based on the above, we project many uses of our novel fibrin-derived microbead technology for cell culturing, wound healing and tissue engineering.

Late effects of dose fractionation on the mechanical properties of breast skin following post-lumpectomy radiotherapy.

PURPOSE: Late radiation-induced skin effects were studied in a multicenter project using our new sensitive noninvasive viscoelasticity skin analyzer (VESA). METHODS AND MATERIALS: Skin viscoelasticity and anisotropy were examined quantitatively in symmetric areas of both breasts in healthy women and in 110 breast cancer patients who underwent lumpectomy and radiotherapy. These parameters were evaluated by the VESA measurement of the speed of elastic wave propagation in the skin; higher VESA readings correspond to higher skin stiffness. Effect of radiation was estimated by comparison of the data recorded in the irradiated versus nonirradiated breast of the same patient. RESULTS: Skin viscoelasticity and anisotropy were similar in contralateral areas of the breasts in healthy controls as well as in the nonirradiated breasts of the patients. With age, skin viscoelasticity decreased and anisotropy increased similarly in both breasts. Radiotherapy, by a total radiation dose in the range of 45-50 Gy given with 1.8 Gy/fraction (fx) resulted in a similar minor, but still statistically significant, increase of skin stiffness relative to control. The effect was more pronounced when a dose of 50 Gy was given in a higher dose/fraction of 2.5 Gy. CONCLUSION: We found that the increase in dose of radiation per fraction had much more impact on the development of late skin effects than elevation in the total dose given.

Chronic graft-versus-host disease treated with UVB phototherapy.

Chronic graft-versus-host disease (cGVHD) is a major complication of allogeneic bone marrow transplantation. Immunosuppressive treatment regimens carry the potential of causing severe morbidity and mortality, so that additional modes of therapy with fewer side-effects are clearly needed. Five cGVHD patients (sclerodermoid cGVHD in two patients, lichenoid cGVHD in one patient and intraoral cGVHD in two patients), who had not responded to standard immunosuppressive drugs, were treated with adjuvant UVB phototherapy. The patient with lichenoid cGVHD experienced complete clearing of cutaneous lesions, whereas both patients with sclerodermoid cGVHD experienced significant relief of pruritus, but showed no change of the sclerodermoid skin lesions. Intraoral lesions cleared in one patient. The effects of UVB phototherapy were furthermore documented by measurement of skin viscoelasticity and mouth opening. No side-effects were encountered. This preliminary study suggests that UVB phototherapy is useful as an adjuvant therapeutic modality in intraoral and cutaneous lichenoid cGVHD.

Paclitaxel-induced modification of the effects of radiation and alterations in the cell cycle in normal and tumor mammalian cells.

The cytotoxicity of paclitaxel (taxol) is associated mainly with block in G2/M phase, the most radiosensitive phase of the cell cycle. Nevertheless, taxol-induced modification of the effects of radiation may vary from clear sensitization to subadditivity. Therefore, this effect was studied in relation to drug-induced alterations in the distribution of cells in the phases of the cell cycle in tumor cells (EMT-6 and OV-1063) and normal skin fibroblasts. Cell survival was evaluated with two colorimetric assays. The cell cycle was evaluated by FACS analysis of doubly-labeled cells. The radiosensitivity of the different cells studied was similar, apart from the less radiosensitive human fibroblasts. However, their dose- and time-dependent sensitivity to taxol varied significantly. After 24 h exposure of EMT-6 cells to taxol (IC50 approximately 20 nM), the fraction of cells in G2/M phase increased, the fraction in S phase decreased, and the proportion of possibly apoptotic cells with subdiploid and subtetraploid DNA content increased; this coincided with radiosensitization. In OV-1063 cells (IC50 approximately 3 nM), the drug-induced G2/M-phase block was most pronounced, but the combined effect with radiation was merely additive. In human fibroblasts (IC50 approximately 35 nM), a minimal G2/M-phase block with no change in the S phase and a massive elevation of apoptotic cells with subdiploid DNA content was accompanied by a subadditive combined effect with radiation. Six hours of exposure to taxol increased the fraction of cells in S phase in both nonsynchronized and S-phase-synchronized human fibroblasts (G1 phase approximately 65%, S phase approximately 13%). This was accompanied by a pronounced subadditive effect of the combined treatment. However, in G1-phase synchronized human fibroblasts (G1 phase > or =90%, S phase approximately 3%), only the fraction of cells in G2/M phase was slightly elevated, with a merely additive response to the combined treatment. The differences in the response to the combined treatment between slowly and rapidly proliferating cells in relation to modifications in the cell cycle are discussed.

A sensitive noninvasive analysis of Pt in external tissues. Followup of Pt deposition following cisplatin treatment.

Noninvasive analysis of heavy elements in external tissues by diagnostic-x-ray spectrometry (DXS) is presented. Pt can be detected accurately with sensitivity below 1 microgram/g wet weight of tissue. In the present paper the possibility to monitor Pt accumulation and clearance in the external tissues of cancer patients treated with cisplatin [Cis-diamminedichloroplatinum (II)-cDDP] chemotherapy is reported. The DXS method is based on x-ray fluorescence analysis. Heavy elements in the small skin area of interest are analyzed by their excitation with a monochromatic soft x-ray beam of 14.6 KeV. Spectral L lines of heavy metals such as Pt are detected with minimum interference by other elements in the tissues. Skin Pt levels up to about 6 micrograms/g were observed following several courses of cDDP treatment. The Pt seemed to be homogeneously distributed in different skin areas with similar levels in the dermis and epidermis. The rate of clearance of Pt from the skin (50% in about 30 days) was slower by three orders of magnitude than its clearance from plasma. Further studies may use DXS to establish the accurate kinetics of Pt deposition and clearance in tissues of cDDP treated patients, as well as the exact relation between tissue Pt levels and the development of the drug related late complications.

Measurement of breast skin viscoelasticity and a pilot study on the potential radioprotective effect of a zinc-based cream.

Radiation-induced late skin effects were studied in patients with breast cancer in relation to different protocols of fractionated radiotherapy in three different medical centres, in Israel, the UK and the USA. The mechanical properties of skin were evaluated in breasts of healthy volunteers, and non-irradiated and irradiated breasts of patients, using a newly developed viscoelasticity skin analyser (VESA). The increase of the dose of radiation per fraction was found to have more impact on the development of radiation-induced late skin effects than the elevation of the total dose given. In addition, a pilot study on the possible radioprotective effect of external application of a cream containing zinc oxide on radiation-induced early skin changes in patients with breast cancer was initiated. Non-invasive measurement of trace elements and zinc pharmacokinetics in the skin of healthy controls following the application of the zinc oxide cream were performed by unique diagnostic X-ray spectrometry (DXS). Application of the cream, followed by thorough skin cleansing, significantly increased the amount of residual zinc in the skin, but continuous daily treatment did not cause further build-up of the dermal zinc level. The radioprotective effect of the zinc oxide cream on the skin is now being studied.

Escin Chemosensitizes Human Pancreatic Cancer Cells and Inhibits the Nuclear Factor-kappaB Signaling Pathway.

Background. There is an urgent need to develop new treatment strategies and drugs for pancreatic cancer that is highly resistant to radio-chemotherapy. Aesculus hippocastanum (the horse chestnut) known in Chinese medicine as a plant with anti-inflammatory, antiedema, antianalgesic, and antipyretic activities. The main active compound of this plant is Escin (C54H84O23). Objective. To evaluate the effect of Escin alone and combined with chemotherapy on pancreatic cancer cell survival and to unravel mechanism(s) of Escin anticancer activity. Methods. Cell survival was measured by XTT colorimetric assay. Synergistic effect of combined therapy was determined by CalcuSyn software. Cell cycle and induction of apoptosis were evaluated by FACS analysis. Expression of NF- κ B-related proteins (p65, I κ Bα, and p-I κ Bα) and cyclin D was evaluated by western blot analysis. Results. Escin decreased the survival of pancreatic cancer cells with IC50 = 10-20 M. Escin combined with gemcitabine showed only additive effect, while its combination with cisplatin resulted in a significant synergistic cytotoxic effect in Panc-1 cells. High concentrations of Escin induced apoptosis and decreased NF- κ B-related proteins and cyclin D expression. Conclusions. Escin decreased pancreatic cancer cell survival, induced apoptosis, and downregulated NF- κ B signaling pathway. Moreover, Escin sensitized pancreatic cancer cells to chemotherapy. Further translational research is required.

Effect of Chinese herbal therapy on breast cancer adenocarcinoma cell lines.

Despite the widespread use of medicinal herbs to prevent and treat many diseases, including cancer, there are insufficient scientific data on the safety and efficacy of the majority of herbal therapies. The aim of this study was to assess the effect of a unique Chinese herbal therapy (CHT) from controlled manufactured concentrated powders, on an in vitro model of breast cancer. Three breast adenocarcinoma cell lines (MDA-231, MDA-453, T47D) were exposed to CHT for 72 h. Cell viability was assessed by XTT (sodium 3'-[1-(phenylaminocarbonyl)-3, 4-tetra zolium]-bis(4-methoxy-6-nitro) benzene sulphonic acid hydrate) assay. Apoptosis and cell cycle stage were determined by fluorescence-activated cell sorting (FACS) analysis. CHT decreased cell survival in a dose-dependent manner in all tested cell lines. FACS analysis of treated and non-treated T47D cells demonstrated that the inhibitory effect of CHT was associated with an increase in apoptosis. A randomized clinical trial is currently underway to investigate CHT as supplementary therapy for breast cancer patients receiving chemotherapy.

Time to pregnancy and multiple births.

BACKGROUND: Mothers of multiples are alleged to be more fecund than mothers of singletons. Some authors have suggested monitoring twinning rates for assessing temporal changes in a population's reproductive health. METHODS: Using a nested case-control design, we estimated the odds of a multiple birth in relation to fecundity in the US Collaborative Perinatal Project inclusive of 8546 pregnant women who reported a known time-to-pregnancy (TTP) upon enrolment in the cohort, 1959-1966. Case mothers comprised 81 women giving birth to twins/triplets; control mothers comprised 243 women giving birth to singletons matched to case mothers on maternal age at a ratio of 3:1. The odds ratio (OR) for a multiple birth within 6 months of trying adjusting for maternal age and prior pregnancies was estimated using logistic regression. Discrete time Cox regression analysis was also utilized to estimate the fecundability OR. RESULTS: Women with a TTP of 6 months [OR=1.95; 95% confidence interval (95% CI)=1.09-3.51]. Excluding pregnancies after 13+ months resulted in a loss of precision (OR=2.14; 95% CI=0.90-5.04). CONCLUSIONS: These data support higher fecundity among mothers of multiples than mothers of singletons.

Establishment and characterization of a pancreatic carcinoma cell line derived from malignant pleural effusion.

BACKGROUND/AIMS: A novel cell line, designated p34, was developed from the malignant pleural effusion of a patient with carcinoma of pancreas. The objective of this work was to characterize this cell line. METHOD: The in vitro studies included karyotype analysis, immunohistochemistry, XTT cell proliferation assay, analysis of the cell cycle by FACS and cell sensitivity to chemotherapeutic drugs and irradiation. Subcutaneous and intra-spleen inoculations into nude mice were carried out to study the tumorigenicity and the metastatic tendency of this cell line. RESULTS: The p34 cell line showed typical morphological characteristics of epithelial pancreatic tumor cells. The cells were hyperdiploid with a modal number of 48, and had two markers, deletion in the short arm of chromosome 2 and duplication of the short arm of chromosome 8. The doubling time was 16 h. Subcutaneous inoculation of the cells into nude mice yielded 100% tumorigenicity, and intra-spleen inoculation resulted in extensive intra-abdominal spread. The antiproliferative effect of chemotherapy (gemcitabine, cisplatin, taxol and vinorelbine), chemopreventive agents (celecoxib and curcumin) and radiotherapy showed dose-dependent cytotoxicity. CONCLUSIONS: This p34 cell line can be used as a new model for studying various aspects of the biology of human pancreatic cancer and potential treatment approaches for the disease.

Changes in intracellular pH induced by hyperthermia and hypoxia.

The effect of hyperthermia on pH of cells cultivated at varying pH of the medium and under different conditions of oxygenation has been studied. Chinese hamster fibroblasts and pig embryo kidney cells were used. The intracellular pH (pHi) was determined by the microfluorimetric method using fluorescein diacetate. The required temperature (37 degrees C and 41 degrees C) was maintained by a warm stage in combination with a thermochamber. Different conditions of oxygenation were achieved by placing the cells into an open (euoxic) or closed (hypoxic) chamber. Both hyperthermia and hypoxia are shown to result in the acidification of cells. The effect increases with the duration of exposure to the agents used. The decrease of pHi is more pronounced when hyperthermia is combined with hypoxia or when the pH of the medium is low. The results obtained correlate with the magnitude of directly damaging and radiosensitizing effects of hyperthermia being dependent on the environmental conditions.

Plasma platinum elimination in a hemodialysis patient treated with cisplatin.

The pharmacokinetics of intravenously administrated cisplatin (CDDP) were studied in a patient with meduloblastoma receiving regular hemodialysis for chronic renal failure. The patient received four CDDP infusions of 25 mg/m2 in two courses and underwent hemodialysis before each treatment and in intervals of 48 h thereafter with blood sampling at the beginning and the end of each hemodialysis. The data obtained were compared with the pharmacokinetic data from follow-up studies of 19 CDDP treatments in 15 patients suffering from various malignancies with no renal failure where a biexponential pharmacokinetic curve was recorded. The data of platinum (Pt) elimination of the patient receiving hemodialysis resembled the curve of those who had not, but following the second CDDP treatment in each course the peak Pt level in plasma was doubled, with a somewhat slower rate of elimination.

Noninvasive follow-up of platinum pharmacokinetics in the skin of patients on cisplatin chemotherapy.

BACKGROUND: Treatment with high-dose cisdiammine-dichloroplatinum (II) (cisplatin, cDDP) often is associated with late complications, predominated by peripheral neuropathy. Pt deposition in different tissues may play a key role in the induction of many of these effects. Main topics of interest include the relationship between cDDP doses given during treatment and the long-term pharmacokinetics of the drug complexes in normal tissues and blood. Noninvasive examination of Pt in tissues during and after cDDP treatment are needed to clarify these points. METHODS: A novel, high-sensitivity diagnostic x-ray spectrometry (DXS) method was used for the fast, noninvasive analysis of Pt in external tissues of patients with cancer treated with courses of cDDP. The Pt in a small skin area was excited by a monochromatic soft x-ray beam (14.6 KeV) and the spectral L lines emitted from the tissue were detected. A limit of detection below 1 microgram/g wet weight was reached. The pharmacokinetics of Pt in blood was investigated in parallel with the use of high-sensitivity, flameless atomic absorption spectrometry (AAS). RESULTS: Follow-up of Pt concentrations in the skin of patients with cancer by DXS before cDDP treatment, during treatment, and up to 4 months after its completion, showed prolonged Pt deposition that corresponded to the net cumulative doses of the drug. Pt clearance from the skin fitted a monoexponential curve with a half-life of about 30 days. In comparison, the pharmacokinetics of total Pt in plasma showed a much faster, biexponential clearance with half-lives of 41 minutes and 5.2 days, respectively. CONCLUSIONS: The amount of nonspecific Pt deposition in the tissues was found to depend on the total doses administered, the time interval between the courses, and the slow rate of clearance. Noninvasive measurements of tissue Pt levels may serve as a major tool in the evaluation of the induction of late cDDP complications.

Reduction of the systemic toxicity of cisplatin by intra-arterial hepatic route administration for liver malignancies.

Cisplatin (CDDP) administration by the intra-arterial hepatic route (i.a.h.) in patients with primary or metastatic liver malignancies could enhance the anti-tumor activity of the drug and reduce its systemic toxicity. The aim of the present study was to compare Pt pharmacokinetics and the toxicity of the circulating drug after i.a.h. versus intravenous (i.v.) administration. CDDP pharmacokinetics was followed-up in 11 i.a.h. courses given to 7 patients with liver malignancies and compared with 19 i.v. courses in 15 patients with cancer of different origins. The Pt level in blood was monitored by sensitive atomic absorption spectrometry. The dose given was in the range of 25-80 mg/m2/treatment. For analysis and for comparison purposes, the data from both CDDP treatments were normalized to a standard dose of 35 mg/m2. The mean peak Pt level for i.a.h. treatment was found to be about half of the mean peak value for i.v. administration with a similar dose-independent bi-exponential rate of elimination i.a.h. CDDP treatment was relatively well tolerated with no symptoms of either nephro- or neurotoxicity. For in vitro evaluation of peripheral CDDP toxicity, a sensitive ovarian carcinoma cell line, OV-1063, was used. A cytotoxic effect was recorded only within 2 hr following high-dose i.v. CDDP treatment. A substantial fraction of the drug given by the i.a.h. route was found to be extracted by the liver in the first passage, with reduced drug level in the peripheral blood plasma relative to the dose given. This may explain the apparent diminution of side-effects following i.a.h. CDDP treatment.

Combination of cisplatin and radiation in cell culture: effect of duration of exposure to drug and timing of irradiation.

Responses to the combination of cisplatin (CDDP) and radiation in experimental and clinical studies have been reported to vary from high radiosensitization to clear sub-additivity. We examined the combined effect of CDDP with ionizing radiation in both murine mammary adenocarcinoma (EMT-6) and human ovarian carcinoma (OV-1063) cells with special reference to the duration of CDDP exposure and timing of irradiation. Cell survival was measured with a colorimetric assay of cell density. The nature of interaction of cisplatin and radiation was evaluated using isobolograms and a combination index (CI). Exposure of both cell lines to CDDP for 24 hr before irradiation yielded an additive or slightly sub-additive response only if the exposure was extended for a few more hours after irradiation. In EMT-6 cells, the combination of radiation with subsequent continuous as well as short-term (4 to 6 hr) CDDP treatment was found to have a clear sub-additive effect; dose escalation of each modality reduced the additional effect of the other. The sub-additive effect may be explained by a radiation-induced arrest of cells in late S phase, which was dose- and time-dependent. Post-radiation exposure to CDDP further increased the S-phase arrest. In contrast, a 2 hr post-radiation drug exposure resulted in a supra-additive combined effect. Our results stress the crucial role of the timing and the doses of both modalities as well as the duration of post-radiation drug exposure on their combined effect.

pH changes in Chinese hamster cells after gamma-irradiation.

Intracellular pH (pHin) changes after gamma-irradiation of Chinese hamster fibroblasts have been studied by a fluorescence method using the ratio of fluorescence intensities after excitation at 488 and 458 nm and measurement at emission wavelength of 515 nm. Irradiation with doses inducing reproductive death (2.5-20 Gy) causes a pHin shift towards the alkaline region by 0.4-0.5 pH units, but this shift is transient. Irradiation with a 500 Gy dose, inducing interphase death, causes a more pronounced (pHin greater than or equal to 8.0) alkalization of the intracellular medium which is retained for more than 1.5 hours post-irradiation. It is proposed that the observed alkalization of the internal medium of irradiated cells is possibly due to a change in the functional state of mitochondria. These changes are probably one of the causes of interphase cell death after irradiation with high doses.

Haptotactic and growth stimulatory effects of fibrin(ogen) and thrombin on cultured fibroblasts.

We tested the ability of purified, ultraviolet C virally inactivated components of human fibrin sealant (FS) to modulate the chemotaxis, adherence, and proliferation of cultured cells. A fibrin clot formed on a near-confluent layer of human fibroblasts (HFs) recruited cells from the surrounding area. Thrombin (Thr) enhanced HF proliferation by a factor of 1.5 to 1.8, whereas fibrinogen (Fib) exerted only a minimal proliferative effect. We developed a new cell haptotactic/attachment assay by using Thr and Fib covalently bound to Sepharose beads (SBs). The kinetics of cell binding were approximately equivalent for beads coated with either protein. Uncoated SBs or fibrinogen-bound SBs (Fib-SB) pretreated with plasmin did not attract HFs. AlphaThr-SB induced a positive migratory response that was not affected by blocking its proteolytic site, whereas gammaThr-SB elicited no response. X irradiation of HFs at a dose of 6 Gy showed that the migratory response of HF is independent of proliferation, as confirmed by a bromodeoxyuridine uptake assay. Several types of cultured cells (murine fibroblasts, smooth muscle cells, aortic endothelial cells, and murine mammary carcinoma cells) also attached to Fib-SB. By contrast, human keratinocytes, human ovarian carcinoma cells, murine macrophage-like cells, leukemic cells, and murine mast cells did not attach. Our results provide some mechanistic insights into the haptotactic and proliferative effects of Fib and Thr on different cells.