99mTc-MAG3 diuresis renography in differentiating renal obstruction: Using statistical parameters as new quantifiable indices.

OBJECTIVE: The aim of this study was to research, develop and assess the feasibility of using basic statistical parameters derived from renogram, "mean count value (MeanCV) and "median count value (MedianCV)", as novel indices in the diagnosis of renal obstruction through diuresis renography. SUBJECTS AND METHODS: First, we re-digitalized and normalized 132 renograms from 74 patients in order to derive the MeanCV and MedianCV. To improve the performance of the parameters, we extrapolated renograms by a two-compartmental modeling. After that, the cutoff points for diagnosis using each modified parameter were set and the sensitivity and specificity were calculated in order to determine the best variants of MeanCV and MedianCV that could differentiate renal obstruction status into 3 distinct classes - i) unobstructed, ii) slightly obstructed, and iii) heavily obstructed. RESULTS: The modified MeanCV and MedianCV derived from extended renograms predicted the severity of the renal obstruction. The most appropriate variants of MeanCV and MedianCV were found to be the MeanCV50 and the MedianCV60. The cutoff points of MeanCV50 in separating unobstructed and obstructed classes as well as slightly and heavily obstructed classes were 0.50 and 0.72, respectively. The cutoff points of MedianCV60 in separating unobstructed and obstructed classes as well as slightly and heavily obstructed classes were 0.35 and 0.69, respectively. Notably, MeanCV50 and MedianCV60 were not significantly influenced by either age or gender. CONCLUSIONS: The MeanCV50 and the MedianCV60 derived from a renogram could be incorporated with other quantifiable parameters to form a system that could provide a highly accurate diagnosis of renal obstructions.

Comparison of the densities of clonogenic cells from EMT6 fibrosarcoma monolayer cultures, multicell spheroids, and solid tumors in ficoll density gradients.

The densities of viable EMT6 cells grown in vitro as monolayer exponential and plateau-phase cells or as multicell spheroids or in vivo as a solid tumor in BALB/c mice were determined with the use of isopyknic centrifugation in linear Ficoll gradients. Exponential cells banded at a density of 1.069 g/ml, whereas plateau-phase cells appeared at 1.073 g/ml. Cells grown as spheroids were more dense than the monolayer cells and were recovered mainly at 1.081. Solid-tumor cells, separated under the same conditions, banded at 1.080. Narrowing the range of the Ficoll gradient failed to resolve more than one band of cells in the solid-tumor separation. This provides evidence that the density of cells obtained from the spheroids is greater than that of the monolayer cells but agrees well with the density of the solid-tumor cells. Ficoll was demonstrated to be nontoxic to the cells, and plating efficiency assays showed similar cell viabilities between noncentrifuged and centrifuged cells. The plating efficiency of the peak fraction of exponential cells after centrifugation was 68%, and that of plateau-phase cells was 50%. Corresponding figures for the multicell spheroid and solid-tumor cells were 62 and 28%, respectively. The recovery of cells after centrifugation in the Ficoll gradients ranged from 62 to 83%. The effects of cell load and centrifugation time on the density distributions of the EMT6 cells were also investigated.

Quantitative means for differentiating renal obstruction by analysing renography by compartmental modelling of renal fluid flow rate.

OBJECTIVE: The aim of this study was to investigate the accuracy of using a newly developed index, the ratio of urine outflow to renal pelvis volume U/V2 (1/s), in evaluating renal obstruction and determining the severity of obstruction. PATIENTS AND METHODS: A total of 42 patients' renograms (80 kidneys) were studied. Compartmental modelling was used to model the behaviour of tracers flowing through the kidney. The derived model led to the formation of the normalized urine flow rate U/V2. An analysis was carried to test the accuracy of the developed index by comparing the developed model and the clinical evaluation of renograms. The Support Vector Machine algorithm was implemented to predict the renal obstruction status. RESULTS: From the comparison performed between the index and the clinical evaluation from certified experts, it was shown that a higher value of index U/V2 indicated a normal kidney, whereas a lower value indicated an obstructed kidney. The classifier developed could provide a 100% accurate diagnosis of differentiated unobstructed kidneys (42/42) and obstructed kidney (18/18). For further classification of obstructed kidneys, the system grouped the samples into slightly obstructed cases with an accuracy of 100% (9/9) and heavily obstructed cases with an accuracy of 89% (8/9). CONCLUSION: The use of the single parameter U/V2 could produce the diagnosis of renal obstruction with a high level of accuracy. This method has the potential to be used as a benchmark to distinguish the severity level of the renal obstruction.

Activity of a selective inhibitor of nuclear export, selinexor (KPT-330), against AML-initiating cells engrafted into immunosuppressed NSG mice.

Currently available combination chemotherapy for acute myeloid leukemia (AML) often fails to result in long-term remissions, emphasizing the need for novel therapeutic strategies. We reasoned that targeted inhibition of a prominent nuclear exporter, XPO1/CRM1, could eradicate self-renewing leukemia-initiating cells (LICs) whose survival depends on timely XPO1-mediated transport of specific protein and RNA cargoes. Using an immunosuppressed mouse model bearing primary patient-derived AML cells, we demonstrate that selinexor (KPT-330), an oral antagonist of XPO1 that is currently in clinical trials, has strong activity against primary AML cells while sparing normal stem and progenitor cells. Importantly, limiting dilution transplantation assays showed that this cytotoxic activity is not limited to the rapidly proliferating bulk population of leukemic cells but extends to the LICs, whose inherent drug resistance and unrestricted self-renewal capacity has been implicated in the difficulty of curing AML patients with conventional chemotherapy alone.

Comparison of radiosensitization by 41 degrees C hyperthermia during low dose rate irradiation and during pulsed simulated low dose rate irradiation in human glioma cells.

PURPOSE: Long duration mild hyperthermia has been shown to be an effective radiosensitizer when given concurrently with low dose rate irradiation. Pulsed simulated low dose rate (PSLDR) is now being used clinically, and we have set out to determine whether concurrent mild hyperthermia can be an effective radiosensitizer for the PSLDR protocol. MATERIALS AND METHODS: Human glioma cells (U-87MG) were grown to plateau phase and treated in plateau phase in order to minimize cell cycle redistribution during protracted treatments. Low dose rate (LDR) irradiation and 41 degrees C hyperthermia were delivered by having a radium irradiator inside a temperature-controlled incubator. PSLDR was given using a 150 kVp X-ray unit and maintaining the cells at 41 degrees C between irradiations. The duration of irradiation and concurrent heating depended on total dose and extended up to 48 h. RESULTS: When 41 degrees C hyperthermia was given currently with LDR or PSLDR, the thermal enhancement ratios (TER) were about the same if the average dose rate for PSLDR was the same as for LDR. At higher average dose rates for PSLDR the TERs became less. CONCLUSIONS: Our data show that concurrent mild hyperthermia can be an effective sensitizer for PSLDR. This sensitization can be as effective as for LDR if the same average dose rate is used and the TER increases with decreasing dose rate. Thus mild hyperthermia combined with PSLDR may be an effective clinical protocol.

Evidence for adaptive response and implication in pulse-simulated low-dose-rate radiotherapy.

PURPOSE: Pulsed-dose-rate (PDR) brachytherapy as a substitute for continuous low-dose-rate (LDR) brachytherapy has a number of clinical advantages. However, early results show that some cells can exhibit an adaptive response to radiation and in PDR where many pulses are given such an adaptive response may play an important role in the outcome. METHODS AND MATERIALS: Nine human cell lines (two normal fibroblast and seven tumor) were evaluated for an adaptive response. Cells were given either a single adapting dose before a challenge dose or given PDR sequences for which the average dose rate matched the LDR dose rate. Response was assessed using the colony survival assay. RESULTS: Five of the nine cell lines showed an adapting response to single small doses of radiation. Three of these cell lines were further investigated for adapting response to PDR and two of the three lines (one ovarian carcinoma and one glioma) showed an adaptive response which was dependent on pulse size and interval. CONCLUSION: The data show that an adaptive response can occur in human cells and that it can vary among cell lines. In addition, PDR sequences also produced an adaptive response which could have an affect on PDR therapy if such a response is found in tissues.

Cisplatin and low dose rate irradiation in cisplatin resistant and sensitive human glioma cells.

PURPOSE: Human glioma cell lines resistant (U373MGCP) and sensitive (U373MG) to cisplatin were used to evaluate the effect of cisplatin as a sensitizer to low dose rate irradiation (LDRI). METHODS AND MATERIALS: A cisplatin resistant glioma cell line U373MGCP was developed by chronic exposure of parental U373MG cells to cisplatin. Plateau phase cells were treated with cisplatin, high dose rate (HDR) irradiation (1.12 Gy/min), LDRI (0.0088 Gy/min), or cisplatin concurrent with LDRI. Cell survival was determined by the colony forming assay. RESULTS: Both cell lines showed increased resistance to radiation at LDR compared with HDR, with Dose Modifying Factors (DMF at 10% survival level) of 1.7 for U373MG and 2.5 for U373MGCP. The increased LDR sparing effect in the cisplatin resistant U373MGCP cells indicates increased repair proficiency. The resistant cell line showed a fourfold increase in resistance to cisplatin cytotoxicity at the 10% survival level compared with the parental U373MG cells. Cisplatin enhanced the response of both cell lines to LDRI. The DMFs were 1.2, 1.2, and 1.7, respectively, for the sensitive U373MG cell line given 1 microgram/ml, and the resistant cell line given 3 or 6 micrograms/ml cisplatin treatments concurrent with LDRI. CONCLUSIONS: These data show that cisplatin can be an effective sensitizer to LDRI in both cisplatin resistant and sensitive glioma cell lines. However, in the resistant cell line, higher concentrations of cisplatin were necessary to achieve the same level of sensitization as in the sensitive cell line.

Equivalence of pulsed-dose-rate to low-dose-rate irradiation in tumor and normal cell lines.

To determine whether different fractionation schemes could simulate low-dose-rate irradiation, ovarian cells of the carcinoma cell lines A2780s (radiosensitive) and A2780cp (radioresistant) and AG1522 normal human fibroblasts were irradiated in vitro using different fraction sizes and intervals between fractions with an overall average dose rate of 0.53 Gy/h. For the resistant cell line, the three fractionation schemes, 0.53 Gy given every hour, 1.1 Gy every 2 h, and 1.6 Gy every 3 h, were equivalent to low dose rate (0.53 Gy/h). Two larger fraction sizes, 2.1 Gy every 4 h and 3.2 Gy every 6 h, resulted in lower survival than that after low-dose-rate irradiation for the resistant cell line, suggesting incomplete repair of radiation damage due to the larger fraction sizes. The survival for the sensitive cell line was lower at small doses, but then it increased until it was equivalent to that after low-dose-rate irradiation for some fractionation schemes. The sensitive cell line showed equivalence only with the 1.6-Gy fraction every 3 h, although 0.53 Gy every 1 h and 1.1 Gy every 2 h showed equivalence at lower doses. This cell line also showed an adaptive response. The normal cell line showed a sensitization to the pulsed-dose-rate schemes compared to low-dose-rate irradiation. These data indicate that the response to pulsed-dose-rate irradiation is dependent on the cell line and that compared to the response to low-dose-rate irradiation, it shows some equivalence with the resistant carcinoma cell line, an adaptive response with the parental carcinoma cell line, and sensitization with the normal cells. Therefore, further evaluation is required before implementing pulsed-dose-rate irradiation in the clinic.

Comparison of thermoradiosensitization in two human melanoma cell lines and one fibroblast cell line by concurrent mild hyperthermia and low-dose-rate irradiation.

Two human melanoma cell lines, one radioresistant (SK-MEL-3) and one radiosensitive (HT-144), and a normal human fibroblast line (AG1522) were evaluated for thermoradiosensitization of low-dose-rate irradiation by concurrent mild hyperthermia (39-41 degrees C). None of the cell lines expressed chronic thermotolerance during heating at 39-41 degrees C. The SK-MEL-3 cells were the most heat sensitive, while AG1522 and HT-144 cells had the same sensitivity at 39 and 40 degrees C but HT-144 cells were more sensitive at 41 degrees C. All cell lines expressed thermal enhancement of radiosensitivity with heating during irradiation which increased with heating temperature. The SK-MEL-3 cells, which were the most resistant to radiation and demonstrated the greatest repair of sublethal damage (SLD) during low-dose-rate irradiation, had the greatest thermal enhancement of radiosensitivity, while the HT144 cells, which were the most sensitive and expressed little repair of SLD during low-dose-rate irradiation, had the smallest thermal enhancement of radiosensitivity. These data show that concurrent mild hyperthermia during low-dose-rate irradiation may be most efficacious in radiation-resistant tumor cells which express resistance through an enhanced capacity for repair of SLD.

Cell cycle perturbations in cisplatin-sensitive and resistant human ovarian carcinoma cells following treatment with cisplatin and low dose rate irradiation.

PURPOSE: To investigate cell cycle pertubations in plateau-phase human ovarian carcinoma cells following treatment with cisplatin, low dose-rate irradiation (LDRI), or combined cisplatin and LDRI, in order to understand cell cycle mechanisms by which these two treatment modalities interact. METHODS: Human ovarian carcinoma cells sensitive (A2780) and resistant (2780CP) to cisplatin were grown to plateau phase and given protracted cisplatin treatments (A2780 0.7 and 2 microg/ml; 2780CP 5 and 15 microg/ml) and/or LDRI (0.41 cGy/min). Cell cycle distribution following treatment was determined by two-parameter flow cytometry, based on bromodeoxyuridine (BrdU) uptake and DNA content using propidium iodide staining. RESULTS: The cisplatin-sensitive A2780 cells exposed to cisplatin alone for up to 28 h showed depletion of the G1 fraction and accumulation in S-phase, although the percentage of S-phase cells actively incorporating BrdU dropped to almost zero. The cisplatin-resistant 2780CP cells exposed to cisplatin alone showed a G1 arrest when exposed to 15 microg/ml, but not when exposed to 5 microg/ml. LDRI alone caused little cell cycle redistribution different from controls in either cell line. When LDRI was combined with cisplatin, no significant cell cycle redistribution was observed, apart from a decline in the actively incorporating S-phase fraction. CONCLUSIONS: Cisplatin caused A2780 cells to accumulate in nonincorporating S-phase, with no evidence of G1 arrest. Cisplatin-resistant 2780CP cells showed a G1 block when exposed to a high enough cisplatin concentration. This could indicate a mechanism of cisplatin resistance in these cells. LDRI alone or in combination with cisplatin did not result in significant cell cycle redistribution.

Interactions of mild hyperthermia, cisplatin and split dose irradiation in human ovarian carcinoma cells.

PURPOSE: Human ovarian cancer cells were evaluated to determine whether combination treatment with mild hyperthermia and cisplatin could inhibit repair of sublethal radiation damage. MATERIALS AND METHODS: Human ovarian carcinoma cell lines A2780S (parental line) and A2780CP (cisplatin resistant variant) were used in this study. Cisplatin at concentrations of 1 or 3 microg/ml was given concomitantly with 1 h of heating at 40 degrees C either immediately before or after irradiation. Survival was determined using a colony-forming assay. RESULTS: Neither mild hyperthermia nor cisplatin treatments alone affected sublethal damage repair. The combined treatment showed an effect in both cell lines and was treatment sequence-dependent. The effect was greater in the parental cell line. CONCLUSIONS: The data show that combined treatment of cisplatin and hyperthermia may have clinical efficacy at cisplatin concentrations and hyperthermia temperatures which by themselves have little to no effect.

A comparison of hyperthermia cisplatin sensitization in human ovarian carcinoma and glioma cell lines sensitive and resistant to cisplatin treatment.

Two pairs of human tumor cell lines (glioma and ovarian carcinoma (OvCa) each having a parental cell line and cisplatin-resistant variant, were evaluated for (a) cisplatin response, (b) hyperthermia response, and (c) combined hyperthermia and cisplatin response. The two resistant lines had comparable resistant responses while for the parental lines, the OvCa was more sensitive than the glioma to cisplatin doses up to 14 microgram/ml. For the hyperthermia response, the OvCa parental line was more resistant than the variant line at low-temperature hyperthermia (41 degrees C or 42 degrees C) but became more sensitive at high temperature (45 degree C). For the glioma, the parental line was more sensitive to hyperthermia at all temperatures tested. Hyperthermia caused sensitization to cisplatin in all cell lines but was generally greater in the glioma cell lines. In the OvCa system, hyperthermia had a slightly greater sensitizing effect on the resistant cell lines, while in the glioma the opposite was true. The degree of sensitization increased with hyperthermia temperature. In summary, the results showed that there is no cross- resistance for hyperthermia and cisplatin, that the degree of thermal sensitization is not reduced in cisplatin- resistant cell lines, and that cisplatin thermal sensitization is cell-line and temperature dependent. Thus, hyperthermia can effectively improve tumor cell response to cisplatin and may be useful in overcoming resistance to cisplatin.

Concomitant treatment with mild hyperthermia, cisplatin and low dose-rate irradiation in human ovarian cancer cells sensitive and resistant to cisplatin.

Hyperthermia has been shown to be an effective radiation sensitizer. Cisplatin has also been shown to cause radiosensitization. In part, the sensitization is through the inhibition of repair of radiation damage. In this study we have set out to combine low dose-rate irradiation (during which extensive repair occurs) with both cisplatin and hyperthermia to maximize the radiation sensitizing effect. Two human ovarian carcinoma cell lines, one parental (A2780S) and the other a cisplatin resistant derivative (A2780CP) cell line were used in these experiments. Long duration hyperthermia at 40 degrees C was combined with low concentrations of cisplatin (0.5-3 microg/ml) and low dose-rate irradiation (LDRI). The responses to the individual treatments showed that there was cross resistance in the two cell lines for cisplatin and radiation, but for hyperthermia the opposite effect was found. When all treatments were given concurrently the response was greater than the calculated response of all three individual treatments, indicating a synergistic interaction. The effect was greater in the cisplatin resistant cell line. The combination of mild hyperthermia, low dose cisplatin and LDRI are a good combination for potential clinical application. In addition, this could be a good approach to deal with cisplatin resistance.

Repair of potentially lethal damage: its inhibition by hyperthermia in two human melanoma cell lines with different radio- and heat sensitivities.

The effect of hyperthermia on the inhibition of potentially lethal radiation damage repair (PLDR) was measured in two human melanoma cell lines of differing radiosensitivities. The HT144 cell line is radiation sensitive and has an exponential survival curve, while the SK-MEL-3 line is radiation resistant and has a shouldered survival curve. Both cell lines demonstrated a similar level of PLDR at isosurvival level radiation doses of 2.0 and 10 Gy for the HT144 and SK-MEL-3 lines, respectively. Hyperthermia (40-45 degrees C) could inhibit PLDR in both cell lines. This inhibition was greater in HT144 than in SK-MEL-3 at low temperatures (40 degrees C) and was reversed at higher temperatures (45 degrees C). There was also a sequence dependence of PLDR inhibition which showed a greater inhibition for heat before irradiation at 40 degrees C but the reverse for temperatures of 41 degrees, 43 degrees and 45 degrees C. These data show that hyperthermia can be an effective inhibitor of PLDR in both radiosensitive and resistant cell lines and that the degree of inhibition is not related to radiosensitivity.

Sublethal radiation damage repair and its inhibition by hyperthermia in two human melanoma cell lines of different radiosensitivities.

Two human melanoma tumour cell lines, one radiation resistant and one radiation sensitive, were studied for their capacity for repair of sublethal radiation damage (SLDR) and for hyperthermia inhibition of radiation damage. The most resistant cell line demonstrated a survival curve with a large shoulder and a large SLDR capacity. The sensitive cell line was characterized by an exponential survival curve and had less SLDR capacity than the resistant line. Hyperthermia could inhibit SLDR in both cell lines and complete inhibition was achieved at 43 degrees C for 30 min. Hyperthermia was more effective after than before irradiation. The SLDR inhibition was greater in the resistant cell line, showing that hyperthermia may be an effective way to overcome resistance in melanoma, especially when such resistance stems from an increased SLDR capacity. In addition, relatively mild thermal treatments, easily achievable clinically, were sufficient to inhibit SLDR in melanoma cells.

Stepdown hyperthermia in human melanoma cells: effects on protracted mild hyperthermia for survival and DNA polymerase inactivation.

Human melanoma cells have been used to evaluate whether stepdown heating (SDH) could increase the effectiveness of long-duration mild hyperthermia (LDMH). The effects of these treatments were also evaluated on cell survival and DNA polymerase inactivation. Short treatments (30 min) at 43 degrees C did not result in much SDH effect for subsequent protracted heating at 40 degrees C. The effect on DNA polymerases was also very small. However, heating at 44 degrees C for 30 min had a large SDH heating effect on subsequent heating at 40 degrees C and 41 degrees C. The SDH effect occurred mainly in the first 5-10 h of subsequent LDMH and, at longer heating times, the rate of cell killing was reduced. The 44 degrees C SDH effect was also observed on DNA polymerase inactivation. Comparing the degree of cell killing and polymerase inactivation showed a good correlation for the various SDH protocols.

Cisplatin and mild hyperthermia in radiosensitization to low dose rate irradiation in human ovarian carcinoma cells.

Two human ovarian carcinoma cell lines, one parental and the other a cisplatin resistant derivative cell line, were evaluated for the efficacy of combined treatments of mild hyperthermia, cisplatin and low dose rate irradiation (LDRI). The data showed that cisplatin (1 or 3 micrograms/ml for 1 hour) combined with mild hyperthermia (40 degrees C for 1 hour) was effective for radiosensitization to LDRI in the parental cell line whether given before or after irradiation. In the cisplatin resistant variant, however, these treatments had little to no effect on LDRI response. Thus these data show that these treatments could be clinically effective if there is no cisplatin resistance. Our previous study showed that concomitant treatments could be effective in both parental and cisplatin resistant cell line (1).

The response of human breast tumour cell lines with altered polymerase beta levels to cisplatin and radiation.

MCF 7 (human breast carcinoma cells) and mutants transfected with the DNA polymerase beta gene were tested for response to cisplatin, radiation and combined treatments. The transfected cells showed a higher level of polymerase beta activity and were more resistant to radiation and cisplatin compared to the parental cell line. Further studies showed that for isosurvival treatments the mutant cells were more effective in sublethal radiation damage repair compared to the parental line. The combination of cisplatin with radiation showed effective radiosensitization which was less in the mutants compared to the parental line. In addition, the sequence of cisplatin before irradiation was more effective then cisplabn after irradiation. Pre-exposure to low levels of cisplatin for up to 24 h before irradiation showed a small significant adaptive response in one mutant line at 8 h and while similar trends were observed in the parental lines at earlier times they were not significant. In summary our data show that polymerase beta and thus base excision repair may play a role in cellular responses to cisplatin and radiation.

Evaluation of cisplatin response in three radiation resistant and three radiation sensitive cell lines.

The cisplatin response was studied in three radiation sensitive mutant cell lines each paired with normally responding or radiation resistant cell lines of parental or similar origin. The radiation sensitive cell lines V3 (CHO), AT5BI (human fibroblast) and HT144 (melanoma) also showed reduced or lack of ability to repair sublethal radiation damage. Also, V3 did not repair radiation induced DNA double strand breaks and AT5BI was excision repair deficient. None of these cell lines show any cross sensitivity to cisplatin treatment. Instead the response was more like the paired normally radiation responding cell lines, except for AT5BI which showed cisplatin resistance. Thus the mechanisms causing radiation sensitivity in the three cell lines are not involved in cisplatin sensitivity.

DNA topoisomerase I content of a pair of human melanoma cell lines with very different radiosensitivities correlates with their in vitro sensitivities to camptothecin.

In this study, we set out to determine if the differential sensitivities to CPT between a radioresistant (Sk-Mel-3) and a radiosensitive (HT-144) human melanoma cell line, and also between cultures with a different growth phase in each cell line, were related to endogenous differences in cellular transport of CPT or to DNA topo I catalytic activities and content. Cultures of HT-144 and Sk-Mel-3 cells in both the exponential, or plateau (i.e. confluent), phase of growth were compared. Cellular response to CPT was determined by clonogenic survival assay. Drug accumulation and efflux were determined using [3H]CPT. Topo I catalytic activity was determined from the ability of nuclear extracts prepared from the cells to relax supercoiled DNA plasmid. Nuclear extracts of the cells were also used to determine topo I content by western blotting. The significantly enhanced sensitivity of exponential-phase, relative to plateau-phase, cultures of both cell lines was related to an enhanced accumulation of [3H]CPT in one (i.e. HT-144), but not the other, cell line. Thus the higher sensitivity of exponential-phase cultures of HT-144 relative to those of Sk-Mel-3 can at least be partially accounted for on the basis of a relatively higher accumulation. However, a higher accumulation was not the reason why plateau-phase cultures of HT-144 were relatively more sensitive than those of Sk-Mel-3. Although there were no significant differences (at the p < 0.05 level) between the endogenous catalytic activities of topo I extracted from exponential- and plateau phase-cultures of both these cell lines, there was a trend for HT-144 cells to show higher endogenous topo I catalytic activities compared to Sk-Mel-3 cells. In contrast, topo I content was higher in exponential- relative to plateau phase-cultures of both cell lines, and in HT-144 relative to Sk-Mel-3 when cultures in a similar growth phase were compared. The relative differences in sensitivity to CPT observed in vitro between these two cell lines, and also between exponential- and plateau phase-cultures of each cell line, correlates best with topo I content rather than topo I catalytic activity or [3H]CPT transport.