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H K Slocum

Publications and source records attributed to H K Slocum.

At least 37 records · Page 2Linked to original sources
PubMed1994

Cellular heterogeneity in DNA damage and growth inhibition induced by ICI D1694, thymidylate synthase inhibitor, using single cell assays.

Heterogeneity in the response of the HCT-8 (human ileocecal adenocarcinoma) tumor cell line to a new thymidylate synthase inhibitor, ICI D1694, was investigated in terms of induction of DNA single-strand breaks and cytotoxicity, applying the single cell alkaline gel (SCG) electrophoresis assay and the individual colony formation assay (iCFA), respectively. ICI D1694 induced maximal total DNA single-strand breaks 24 hr after a 2-hr drug exposure with incomplete repair by 72 hr. The level of DNA damage was concentration dependent and paralleled cellular growth inhibition in vitro. The proportion of cells with DNA damage and the extent of DNA single-strand breaks increased with drug concentration. At 1 microM ICI D1694 (IC95), a significant level of DNA damage was detected in 58% of the cells; however, 25% of the cells had little or no damage. Using the iCFA system, it was observed that with 1 microM ICI D1694, only 2.6% of the seeded cells maintained a colony growth rate similar to that of the control colonies, and 22% of the cells were growing significantly more slowly. In conclusion, the SCG assay and the iCFA identified subpopulations of cells that were unaffected by ICI D1694. Although these cells represented only a small proportion of the total cell population, this phenomenon of heterogeneity in response to ICI D1694 might limit its therapeutic efficacy.

Cell Division↗
PubMed1994

New immunohistochemical "sandwich" staining method for mdr1 P-glycoprotein detection with JSB-1 monoclonal antibody in formalin-fixed, paraffin-embedded human tissues.

We have developed a new immunoperoxidase "sandwich" staining method for amplified detection of P-glycoprotein (Pgp) that is suitable for use on formalin-fixed, paraffin-embedded (conventional) tissue sections. This was accomplished by substantially changing the procedure described by Chan (1988) so as to increase specific staining intensity and to decrease nonspecific background staining. To determine the most appropriate primary antibody for the assay, we compared the immunoreactivity of JSB-1, C494, and C219 monoclonal antibodies recognizing internal epitopes of Pgp, and MRK16 and 4E3 monoclonal antibodies recognizing external epitopes of Pgp. Paraffin sections of Pgp-positive normal human tissues (adrenal, liver, kidney, and brain), of renal tumors, and of cell pellets of sensitive and multidrug resistant human tumor cell lines (MCF-7, KB) were used for comparisons. Immunostaining was excellent with JSB-1, moderate with C494, and very weak with C219. MRK16 and 4E3 showed no reaction. Nonspecific background staining was reduced by 1) omitting immunoglobulin G from secondary antibodies; 2) decreasing the concentration of peroxidase-antiperoxidase complex; and 3) utilizing casein solution for blocking and washing. Pretreatment of sections before immunostaining was also simplified. Using JSB-1, the threshold for detection of elevated Pgp corresponded to less than two-fold relative resistance to doxorubicin. Applying this method, we found two of 26 non-small cell lung cancers were positive for Pgp, consistent with previous results of others using frozen sections. This new immunoperoxidase sandwich staining method using JSB-1 now allows reliable Pgp detection in sections of formalin-fixed, paraffin-embedded (archived) surgical specimens and small biopsy materials commonly used for diagnostic purposes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Isolation and characterization of a human ileocecal carcinoma cell line (HCT-8) subclone resistant to fluorodeoxyuridine.

A 5-fluoro-2'-deoxyuridine (FdUrd)-resistant subclone (Fd9XR) of HCT-8 (human ileocecal carcinoma) cells was established by two schedules of drug exposure. Initially, cells were exposed to short-term (3 hr) 100 nM FdUrd repeatedly (9 cycles over 8 months), and cells were then exposed to 10 nM FdUrd continuously. During this latter stage, a colony (Fd9XR) with fast growth rate was isolated, expanded, and characterized with respect to mechanisms of resistance to FdUrd and cross-resistance to other chemotherapeutic agents. Fd9XR cells were 1000-fold resistant to FdURD, but 3-fold more sensitive to 5-fluorouracil (FUra) than HCT-8 cells. After a 3-hr treatment with FdUrd, Fd9XR cells accumulated 6630-, 69-, and 3.7-fold less fluorodeoxyuridylate (FdUMP), fluorouridine triphosphate (FUTP) and acid-insoluble materials, respectively, than HCT-8 cells. However, when FUra was substituted for FdUrd, Fd9XR cells accumulated 9.2-, 3.1-, and 2.3-fold more FdUMP, FUTP and acid-insoluble materials, respectively, than HCT-8 cells. Fd9XR and HCT-8 were similar in their growth rates, combined pools of 5,10-methylenetetrahydrofolates (5,10-CH2H4PteGlun) and tetrahydrofolates (H4PTeGlun), thymidine phosphorylase (TP) activity, and level and activity of thymidylate synthase (TS). In contrast, thymidine kinase (TK) activity of Fd9XR was 0.23 and 0.35% of that of HCT-8, for thymidine (dThd) and FdUrd as substrates, respectively. Furthermore, Fd9XR cells exhibited greater sensitivity to the antifolate TS inhibitor ICI D1694 and to methotrexate (MTX) than HCT-8 cells. In addition, dThd alone and in combination with hypoxanthine did not offer any protection against the cytotoxic effect of ICI D1694 in Fd9XR cells. These results indicate that in Fd9XR cells (1) TK deficiency is the primary mechanism of resistance to FdUrd; (2) the greater sensitivity to FUra was associated with higher pools of FdUMP and FUTP with a subsequently higher level of incorporation into cellular RNA; and (3) antifolate compounds, e.g. ICI D1694 and MTX, could be useful agents in the treatment of FdUrd-resistant tumors associated with decreased TK activity and decreased capacity of utilizing dThd.

Antimetabolites, Antineoplastic↗
PubMed1993

Growth slow-down and growth arrest of human colon carcinoma cells HCT-8 in vitro after exposure to 5-fluoro-2'-deoxyuridine.

Cellular heterogeneity in drug response denotes a mixed response among individual cells in a drug treated population. Individual cell responses may be more complex than 'cell kill' and 'no response'. In this study we employed a colony formation assay and high-resolution image analysis to detect the various responses such as immediate and delayed cessation of growth, growth delay and growth slow-down, at the level of the individual colony. The evaluation was carried out using a human ileocaecal adenocarcinoma cell line (HCT-8) and the anti-tumour agent 5-fluoro-2'-deoxyuridine (FdUrd). In the presence of a drug concentration which, in standard monolayer assays, inhibits the growth to about 50% (IC50) only about 20% of the colonies ceased to grow and the remaining colonies grew at a growth rate of about 70% of control. At an FdUrd concentration which, in standard monolayer assays, reduced growth by > 90% (> IC90), about 50% of the cells grew, with growth rates of about 30% of control. The slowing of growth, most prominent at lower drug concentrations, should be considered in determining mechanisms of drug action at the individual cell level. In clinical situations in which high drug doses are precluded by toxicity to normal tissues, growth slow-down may play a significant role in tumour response.

Cell Division↗
PubMed1993

Effect of doxorubicin, daunorubicin, and idarubicin on the growth rate of human leukemia cells (K562) studied with image analysis.

The effects of doxorubicin, daunorubicin, and idarubicin on the growth of K562 cells were investigated by monitoring the formation of individual colonies in semi-solid culture employing an automated image analysis system. Drug effects were evaluated using short-term exposure (2 h) at clinically achievable drug plasma concentrations. Following drug exposure, heterogeneity in growth patterns was observed which was categorized into three distinct types; (1) continuous growth at a decreased growth rate; (2) limited growth; (3) no growth. In addition to a concentration-dependent decrease of the growth fraction, a reduction of the growth rate of the continuously growing colonies was observed. At equitoxic drug concentrations, comparable changes in growth rate were observed for each of the three anthracyclines studied. It is demonstrated that the fraction of cells which escape drug-induced growth arrest with therapeutically achievable drug concentrations display a significant decrease in growth rate.

Cell Division↗
PubMed1993

Characterization of tumor cell heterogeneity of a murine leukemia cell line (L1210) in response to arabinosylcytosine: quantitation using a computerized image analysis system.

Cytosine arabinoside (Ara-C) is one of the most effective drugs in inducing remission in acute nonlymphocytic leukemia (ANLL) patients. However, the high recurrence rate indicates that a subpopulation of leukemic cells escapes drug effect. This cellular heterogeneity in drug response may play a major role in chemotherapeutic outcome. We have recently developed the individual colony-formation assay (ICFA) to study drug effects on the kinetics of proliferation of individual cells and their progeny. Thus parameters of proliferation are calculated for individual colonies. Three categories of drug responses were defined, including immediate growth cessation, delayed growth cessation (growth stops several days after drug exposure) and growth slowdown (logarithmic growth at a reduced rate compared to control). In the experiments included in this report, murine leukemia (L1210) cells were exposed to various concentrations of Ara-C for 1, 6 or 24 hours, and their responses quantified. Regardless of the Ara-C concentration or exposure time, subpopulations of cells were observed in each of the three response categories: immediate or delayed arrest or growth slowdown. As expected, the fraction of cells exhibiting immediate growth cessation generally increased with increasing drug dose and was markedly increased with longer exposure time. Delayed arrest was most prevalent at intermediate drug concentrations at all exposure times. If exposure was limited to 1 hour, at least 30% of cells continued to grow, although at a reduced rate (71% control rate after exposure to 1 mM Ara-C). This limited effect was paralleled by saturation of Ara-C triphosphate (Ara-CTP) formation. Six-hour exposure left at least 6.4% of cells growing, with an average rate of 45% of control. Under these conditions, no saturation in Ara-CTP formation was observed. Even 24-hour exposure to 5 microM Ara-C left 4.8% of colonies growing, at 42% of control rate. Thus a subpopulation of cells continued to grow even after 24-hour exposure to a relatively high concentration of Ara-C. Surviving, but slowly growing, cells may represent a previously unrecognized population that may contribute to therapeutic failure.

Animals↗
PubMed1992

Growth of human renal cortical tissue on collagen gel.

A model system for 3-dimensional "native-state" culture of tissues on collagen gels (Proc. Natl. Acad. Sci. USA 86:2013-2017; 1989) has been applied in this study to histologically normal human renal cortical tissue from 11 patients undergoing nephrectomy for renal cell carcinoma elsewhere in the kidney. Microbial contamination occurred in 12/90 cultures, the rest (78) were studied by visual inspection, histology, immunohistochemical analysis for pankeratin (epithelial cell origin), vimentin (mesenchymal cell origin), and p-glycoprotein (associated with proximal tubules), transmission electron microscopy (EM), incorporation of tritiated thymidine (3HTdR). In the first 10 days, explants showed 3HTdR-labeled cells in tubule structures. The surrounding gel was invaded by cells forming tubule structures, sometimes with basement membrane. Some of these cells showed labeling by 3HTdR and immunostaining positive for pankeratin and p-glycoprotein. EM showed well-polarized epithelial cells in tubule structures with tight junctions, interdigitating lateral processes, and microvilli characteristic of proximal and distal convoluted tubules. 3HTdR-labeled cells in tubule structures were observed even 2 mo. after Passage 1, 6 mo. after the initial explantation. Tubule growth was most active and fibroblast proliferation was negligible from 2 to 4 wk postexplantation. The proliferation of tubulelike cells and formation of tubulelike structures in this system represents an opportunity to study human renal cortical tissue in vitro, under conditions more closely resembling in vivo circumstances than are present in other in vitro systems suitable for long-term study. This model has potential use for in vitro toxicology studies and studies of renal physiology.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Comparison of an immunoperoxidase "sandwich" staining method and western blot detection of P-glycoprotein in human cell lines and sarcomas.

The applicability of a multilayer immunoperoxidase "sandwich" method (IpS) developed by Chan14 for the amplified detection of P-glycoprotein (Pgp) was investigated. The authors examined 15 formalin-fixed cell lines, as well as formalin-fixed, paraffin-embedded sections from single biopsies of 46 sarcomas. The cell lines included sensitive and multidrug resistant sublines (KB, A2780, MCF-7, HeLa) with various relative degrees of resistance to doxorubicin (Dox). The sarcoma biopsy specimens were selected on the basis of the results obtained in Western blot (WB) detection of Pgp (22 positive and 24 negative by WB) using C219 and C494 monoclonal antibodies to Pgp. The IpS method employed C219. The least resistant cell line in which Pgp could be detected by IpS was fivefold resistant to doxorubicin, whereas Pgp was detected by WB in cells greater than twofold resistant. Cell lines having greater than fivefold resistance to Dox were positive by both IpS and WB analyses. The less resistant cell lines contained more nonreactive cells whereas the highly resistant cell lines showed more homogeneous strong membrane reactions. Among the six cell lines determined to be Pgp negative by WB analysis, no false positive immunostaining by IpS existed. One of 22 WB positive and 7 of 24 WB-negative sarcoma biopsy specimens were positive by IpS methods. Reaction varied and was always focal (a minimum of 3-5 cells, ranging up to 3-4 high power fields) indicating pronounced heterogeneous distribution of Pgp. Thus, WB can detect low average (overall) levels of Pgp in tumor samples but such low concentrations of PgP at the single cell are not detectable by IpS methods. However, IpS can discern among many Pgp-negative cells small subpopulations of immunoreactive cells, which are not detected by WB analysis due to Pgp dilution by the membrane protein of numerous Pgp negative cells. IpS and WB used together as complementary methods can provide more complete information about Pgp distribution and content, particularly in the case of heterogeneous human tumors. The IpS method is more suitable for less drastically treated (not embedded) cell line specimens than for paraffin-embedded (routine) sections. Some modification of the present IpS protocol seems necessary to increase its sensitivity and reduce the disparity with WB results.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

In vitro study of the interaction of doxorubicin, thiotepa, and mitomycin-C, agents used for intravesical chemotherapy of superficial bladder cancer.

Several cytotoxic agents have been identified as effective in the treatment of superficial transitional cell carcinoma of the bladder, including doxorubicin, thiotepa and mitomycin-C. An in vitro study was conducted to assess the interactions of these three drugs against a well differentiated human bladder tumor cell line, RT-4, to identify and evaluate synergistic combinations among these agents. Cytotoxicity was evaluated by a colorimetric assay based on the capacity of viable cells to metabolize a tetrazolium dye, MTT, to produce a colored formazan product. The analyses of drug interactions were done by the isobolographic method (construction of isoeffect plots). The combination of doxorubicin and thiotepa was found to be the most synergistic, followed by the combination of doxorubicin and mitomycin-C. The combination of mitomycin C and thiotepa demonstrated an unpredictable effect. These findings suggest the combination of doxorubicin and thiotepa has potential advantage for chemotherapy of superficial bladder tumors.

Antineoplastic Agents↗
PubMed1991

A model for mimicking the pharmacokinetics of chemotherapy drugs for evaluation of drug effects in a soft agar colony formation assay system.

Colony formation assay systems are an important part of in vitro drug evaluation. It would be useful if the in vitro drug cytotoxicity could be carried out under conditions mimicking those employed clinically. We have developed an individual colony formation assay system that would allow monitoring and quantitation of the growth of individual colonies (9) in the presence of the drug under conditions of continuous and/or short-term exposure, after plating of the cells in the agarose. In this brief report, we established the pharmacokinetic profile of four drugs, cytosine arabinoside (araC), Doxorubicin (Dox), 5-fluorouracil (5-FUra) and 5-fluoro-2'-deoxyuridine (FdUrd) in agarose mimicking those that are achieved when these agents were administered in vivo as an i.v. push. The results show that short-term treatment in agarose is possible and that the washing procedure of the drugs decreased the drug concentrations 3-4 logs over 44 hours.

Antineoplastic Agents↗
PubMed1990

The determination of growth rates of individual colonies in agarose using high-resolution automated image analysis.

This paper describes the evaluation of a colony formation assay using automated image analysis, which permits the tracking of growth at the individual colony level, such that a growth rate can be estimated for each colony followed. In principle, this will permit quantitative characterization of cellular heterogeneity in growth rate and cellular heterogeneity in response to proliferation-modifying agents. In addition, we have demonstrated the possibility of using correlative microscopy to relate growth rate to other parameters, using metabolic viability as an example. This should be useful for determining cellular characteristics associated with proliferative behavior and response to proliferation-modifying agents.

Adenocarcinoma↗
PubMed1990

Assessment of human genitourinary tumors and chemosensitivity testing in 3-dimensional collagen gel culture.

A recently described collagen gel culture technique has been modified to evaluate the growth characteristics and chemosensitivity patterns of genitourinary neoplasms. Fresh human surgical explants incorporated radiolabeled DNA precursors [H3)thymidine or deoxyuridine) in 97% of 38 patient specimens (18/19 bladder, 3/3 prostate, 2/2 testis, 13/14 renal), after being maintained for three to 13 weeks/passage, with several specimens reaching their sixth passage (20 months). Control cellular DNA incorporation ranged from five to 90% (#cells labeled/#cells evaluated), with median labeling for bladder 30%, prostate 80%, testis 90%, and renal 80%. Original histopathologic classification was maintained in all cases. Tumor volume and glucose consumption were other measurable parameters. Seventy-three surgical specimen cultures were treated with chemotherapeutic agents after a minimum of four weeks in culture. Single agent exposures were 24 hours at 1X and 10X reported peak plasma concentrations. Combination agents were sequenced as in current clinical protocol for bladder tumors and fourteen day continuous fluorodeoxyuridine (FdURD) exposure for renal tumors. Sensitivity was found in 1/2 prostate and 1/10 renal tumors to Adriamycin, 8/15 bladder and 1/2 testis tumors to cisplatin, 11/28 renal tumors to FdURD and 6/16 bladder tumors to MVAC combination chemotherapy. This culture system offers the advantages of in vivo-like solid tumor growth, a high culture success rate, longevity in culture, maintenance of the primary histopathology and reproducible chemosensitivity response.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1989

Inhibition of implantation of murine bladder tumor by thiotepa in cauterized bladder.

This study was designed to determine the role of immediate intravesical instillation of single dose thiotepa post transurethral resection of bladder tumor in the prevention of recurrence by tumor implantation, using murine bladder tumor line 2 and 201 C3H/He mice. Previous studies have suggested implantation may take place as early as the first hour and reach its maximum in 24 hours after resection of bladder tumor. An in vitro dose response curve of MBT2 to thiotepa was established by treatment with various concentrations of thiotepa of 0.00, 0.01, 0.21, 0.44, and 1.91 mg./ml. In a group of 201 mice, the bladder was catheterized with a 24G angiocatheter, and a fine copper wire was inserted through the lumen. The bladder was cauterized by touching the wire with a Bovie coagulator for four seconds at the lowest setting. All bladders were instilled with 1 x 10(6) cells of murine bladder tumor line 2, followed by instillation of 1.91 mg./ml. of thiotepa with various time delays per treatment group. The bladder implantation rates were 30.4% (17/56), 3.4% (2/59), 6.5% (2/31) and 26.9% (7/26) in the control, immediate, one-hour delay and 24-hour delay groups, respectively. The urethral implantation rates were 21.4% (12/56), 0% (0/59), 6.5% (2/31) and 0% (2/26), respectively. The overall implantation rates (bladder, urethra, or both) were 42.9% (24/56), 3.4% (2/59), 6.5% (2/31) and 25.9% (7/27), respectively. Implantation rates were significantly higher in the control and 24-hour delay groups than in the immediate and one-hour instillation groups (p less than 0.05, Fisher Exact Test). We conclude from this animal model that intravesical instillation of single dose thiotepa, to be effective, should be initiated within the first hour after tumor resection, since it dramatically decreased the incidence of bladder and urethral implantation.

Administration, Intravesical↗
PubMed1987

Biological activities of 5-fluorouracil and its prodrug 5'-deoxy-5-fluorouridine in rats.

The antitumor activity and toxicity of 5-fluorouracil (FUra) and 5'-deoxy-5-fluorouridine (dFUR) were compared in female Fischer rats bearing transplanted dimethylhydrazine-induced colon tumors. The therapeutic effects of FUra and dFUR were not affected by the initial tumor size, but were dependent on the dose and duration of treatment. The maximal response rate of 80-90% cures was obtained with 7-day infusions of 35 mg-kg-1-day-1 FUra or 500 mg-kg-1-day-1 dFUR. The host toxicity of FUra and dFUR in tumor-bearing or normal rats included gastrointestinal and central nervous system disturbances. Toxicity related death was preceded by a greater than 20% animal weight loss and other signs of gastrointestinal disturbances. The maximal therapeutic dose of FUra was identical to the toxic dose which caused 40% death in normal rats. By contrast, the maximal therapeutic dose of dFUR did not cause toxic death, and the threshold lethal dose of dFUR was 40% higher than the maximally therapeutic dose, indicating a better therapeutic index for dFUR in this rat tumor.

Animals↗
PubMed1987

P-glycoprotein in human sarcoma: evidence for multidrug resistance.

Overexpression of an immunologically conserved, cell-surface glycoprotein (P-glycoprotein) is consistently associated with multidrug resistance in cell lines in vitro. A preliminary survey of specimens from 12 solid tumor types in our laboratories indicates significant overexpression of P-glycoprotein in some sarcomas. When tested by immunoblotting with monoclonal antibodies directed against P-glycoprotein; tumors from six of 25 sarcoma patients displayed elevated levels of P-glycoprotein. Three of the sarcoma patients exhibiting P-glycoprotein had not previously been exposed to chemotherapy, implying that overexpression of this marker and possible concomitant multidrug resistance may not depend only on selection during prior drug treatments. The P-glycoprotein overexpression in the sarcoma specimens is evidence for the presence of multidrug resistant cells in these tumors; thus, our data suggest that this mode of resistance may have clinical significance in sarcoma patients.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1986

Growth of human urologic tumors on extracellular matrix.

Surgical specimens of fifty urologic tumors, 19 renal, 19 bladder, nine prostate and three testicular, were disaggregated into a cell suspension by a two step mechanical and enzymatic method. Viability, cytology and flow cytometry (FCM) for DNA ploidy were subsequently determined. Growth of urological tumors on extracellular matrix (ECM) was carried out as follows: 2.5 to 5 X 10(5) cells were placed in plastic T25 flasks in RPMI 1640 + 10 per cent fetal bovine serum (FBS). 1.0 to 5 X 10(4) cells were plated in wells coated with ECM derived from bovine corneal endothelial cells with RPMI 1640 + 10 per cent FBS. Cultures were incubated for seven to 10 days at 37 degrees C. Only one (renal) out of 28 tumors grew on plastic. Forty out of 50 tumors (80 per cent) established primary cultures on ECM as determined by cell counting, protein determination, and/or [3H]thymidine incorporation. Previous experience with 106 urologic tumors grown on double layer agar demonstrated an overall success rate of 48 per cent. On ECM renal tumors showed 95 per cent growth success, prostate 89 per cent, bladder 63 per cent and testicular 67 per cent. Unlike viability by trypan blue exclusion, tumor DNA ploidy and percentage of malignant cells plated on ECM had no effect on growth success. The malignant nature of the cultured cells was confirmed by cytology. Twelve high grade and metastatic tumors caused degradation of the ECM. DNA ploidy was similar in four and different in six tumors before and after culture. Five tumors underwent in vitro drug testing on ECM with significant growth inhibition observed in three cases. The extracellular matrix seems to be a promising model for growing urologic tumors with excellent potential for drug testing in vitro.

Antineoplastic Agents↗