Adjustment for body mass index and calcitrophic hormone levels improves the diagnostic accuracy of the spot urine calcium-to-creatinine ratio.

SUMMARY: Providers diagnose hypercalciuria using a 24-hour or random urine samples. We compared calcium measurements from paired 24-hour and morning urine samples; measurements correlated poorly. We developed a formula to correct random urine calcium levels. Corrected levels showed excellent agreement with 24-hour measurements. Until validation, providers should diagnose hypercalciuria using 24-hour tests. INTRODUCTION: Hypercalciuria is a risk factor for osteoporosis and nephrolithiasis. The 24-hour urine calcium (24HUC) measurement is the gold standard to diagnose hypercalciuria, but the spot urine calcium-to-creatinine ratio (SUCCR) is more convenient. Although authors claim they are interchangeable, we observed inconsistencies during the conduct of a clinical trial. Therefore, we systematically evaluated agreement between the tests. METHODS: During a 28-inpatient calcium absorption studies in 16 postmenopausal women, we simultaneously collected paired fasting morning and 24-hour urine specimens. RESULTS: We found moderate correlation between paired SUCCR and 24HUC specimens (r = 0.57, p = 0.002), but the SUCCR underestimated 24HUC by a mean of 83 mg (Bland-Altman). We diagnosed hypercalciuria (24HUC >250 mg) in eight specimens using the 24HUC, but only in two specimens using the SUCCR (25% sensitivity). We developed a regression model to predict 24HUC using SUCCR, parathyroid hormone, body mass index, and 1,25(OH)(2)D. The model improved diagnostic sensitivity to 100% and decreased Bland-Altman bias of the SUCCR to +0.06 mg/kg/24-hour. CONCLUSIONS: We conclude that the SUCCR underestimates urine calcium loss and does not reliably diagnose hypercalciuria. A formula derived from multivariate regression incorporating other readily measurable variables greatly improved the SUCCR's accuracy. Future studies must verify this correction before clinical implementation.

Herbicide sorption coefficients in relation to soil properties and terrain attributes on a cultivated prairie.

The sorption of 2,4-D and glyphosate herbicides in soil was quantified for 287 surface soils (0-15 cm) collected in a 10 x 10 m grid across a heavily eroded, undulating, calcareous prairie landscape. Other variables that were determined included soil carbonate content, soil pH, soil organic carbon content (SOC), soil texture, soil loss or gain by tillage and water erosion, and selected terrain attributes and landform segments. The 2,4-D sorption coefficient (Kd) was significantly associated with soil carbonate content (-0.66; P < 0.001), soil pH (-0.63; P < 0.001), and SOC (0.47; P < 0.001). Upper slopes were strongly eroded and thus had a significantly greater soil carbonate content and less SOC compared with lower slopes that were in soil accumulation zones. The 2,4-D Kd was almost twice as small in upper slopes than in lower slopes. The 2,4-D Kd was also significantly associated with nine terrain attributes, particularly with compounded topographic index (0.59; P < 0.001), gradient (-0.48; P < 0.001), mean curvature (-0.43; P < 0.001), and plan curvature (-0.42 P < 0.001). Regression equations were generated to estimate herbicide sorption in soils. The predicted power of these equations increased for 2,4-D when selected terrain attributes were combined with soil properties. In contrast, the variation of glyphosate sorption across the field was much less dependent on our measured soil properties and calculated terrain attributes. We conclude that the integration of terrain attributes or landform segments in pesticide fate modeling is more advantageous for herbicides such as 2,4-D, whose sorption to soil is weak and influenced by subtle changes in soil properties, than for herbicides such as glyphosate that are strongly bound to soil regardless of soil properties.

Limonene-induced regression of mammary carcinomas.

Dietary administration of the monocyclic monoterpenoid d-limonene causes complete regression of both dimethylbenz[alpha]anthracene- and N-nitroso-N-methylurea-induced rat mammary carcinomas. Carcinomas regress when limonene is added to the diet either when the tumor is small and still capable of spontaneously regressing or when it is large and progressed beyond the stage when it is susceptible to spontaneous regression. The limonene dose-tumor regression response relationship is steep. Significant regressions are not observed at 5% dietary levels, while a majority of tumors completely regress above a 7.5% dietary level. Limonene appears to act in a cytostatic fashion. Its removal from the diet results in a significant number of tumor recurrences. Regressing tumors have a unique histopathological appearance that is not associated with gross cytotoxicity, immune cell involvement, or apoptosis. Preliminary analysis suggests a remodeling/redifferentiation event underlying regression. The underlying mechanism of action of limonene in causing tumor regression is unknown. However, it should be noted that limonene can selectively inhibit the isoprenylation of small G proteins. Monoterpenoids such as limonene represent a novel class of anticancer drugs with the potential to cause tumor regressions with limited toxicity.

The interaction of hydroxyurea and iododeoxyuridine on the radiosensitivity of human bladder cancer cells.

Biochemical modulation of iododeoxyuridine (IdUrd) incorporation into the DNA of tumor cells is a potential clinical strategy to enhance radiosensitivity and to simultaneously differentiate the sensitivity of rapidly proliferating tumor cells and more slowly proliferating adjacent normal tissues to radiation. The interactions of hydroxyurea (HU) and IdUrd were studied in a human bladder cancer cell line, 647V. Exposure of exponentially growing 647V cells to HU concentrations of 10-100 microM for one cell population doubling (24 h) resulted in no cytotoxicity as assessed by clonogenic survival. Flow cytometric analysis showed a significant increase in an early S-phase population after a 12-h exposure but a return to a normal cell cycle distribution after a 24-h exposure to 100 microM HU. Incorporation of IdUrd into DNA was increased 2-fold by coincubation with HU (100 microM) and a clinically achievable concentration of IdUrd (2 microM) for 24 h. To elucidate the mechanism of modulation, IdUTP pools were compared in 647V cells treated with 2 microM IdUrd with or without 100 microM HU. A 2-fold increase in IdUTP pools was evident within 2 h when this drug combination was used. With the use of multivariate statistical analysis, the radiosensitivity of 647V cells was compared after a 24-h exposure to various concentrations of IdUrd (0 and 2 microM) and HU (0, 10, and 100 microM). A 24-h exposure to 100 microM HU alone or to 2 microM IdUrd alone before irradiation resulted in significant (P < 0.02) radiosensitization with sensitizer enhancement ratios of 1.15 and 1.27, respectively. A 24-h exposure to 100 microM HU + 2 microM IdUrd resulted in even more significant (P = 0.001) radiosensitization, which was found to be a greater than additive response (sensitizer enhancement ratio, 1.76 observed compared with 1.37 expected). No radiosensitization was found with a 12-h exposure to 100 microM HU alone. The mechanism of biochemical modulation of IdUrd by a noncytotoxic dose of HU is proposed as increasing the IdUTP pools by stimulating enzymes in the thymidine salvage pathway and subsequently enhancing IdUrd incorporation and radiosensitization.

Carcinoma induction following direct in situ transfer of v-Ha-ras into rat mammary epithelial cells using replication-defective retrovirus vectors.

Chemically induced mammary carcinomas often contain the activated Ha-ras oncogene. The role of this oncogene in the multistage process of carcinogenesis remains undefined. In order to model the role of ras in mammary carcinogenesis, gene transfer into adult rat mammary epithelial cells was accomplished by infusing helper-free, replication-defective retrovirus vectors into the central duct of each gland. In the initial experiments, the beta-galactosidase reporter gene was used to optimize the efficiency of this in situ gene transfer method. Stable infection of greater than 0.1% of mammary cells could be achieved following exposure to the beta-galactosidase gene-expressing vector. v-Ha-ras was then introduced into in situ adult rat mammary epithelial cells using this method. Cellular infection frequencies of less than 1% resulted in the frequent and rapid appearance of mammary carcinomas without any further treatment. Tumors arising following v-Ha-ras oncogene transfer resembled those induced by chemical carcinogens in both the kinetics of their development and histopathological spectrum. These observations support the hypothesis that ras activation can act as an initiation event in chemically induced mammary carcinogenesis. However, only a small percentage of v-Ha-ras infected cells, even with hormonal promotion, were neoplastically transformed, suggesting that ras-driven transformation is not a one-step event.

Overcoming the activity of mammary carcinoma suppressor gene in Copenhagen rats by v-H-ras oncogene transfer into mammary epithelial cells in situ.

Susceptibility to mammary cancer in rats is genetically controlled by both susceptibility and suppressor genes. The Copenhagen (COP) rat strain is highly resistant to both spontaneous and induced mammary carcinogenesis. The resistant trait is due to the inheritance of an autosomal dominant allele termed mammary carcinoma suppressor (mcs) gene. To test whether the activity of mcs gene can suppress the transforming potential of an activated oncogene, we introduced v-H-ras oncogene into COP mammary epithelial cells in situ using a replication-defective retroviral vector. v-H-ras transfer caused the rapid development of mammary carcinomas at high multiplicities. Hormonal promotion further increased the penetrance of the activated ras gene. Compared with the mammary carcinoma-susceptible Wistar Furth (WF) rat strain, tumor development in the COP rat followed analogous kinetics. However, COP tumors were more differentiated and less locally invasive than were WF tumors. The possible role of the mcs gene in mammary differentiation is discussed.

Frequent induction of mammary carcinomas following neu oncogene transfer into in situ mammary epithelial cells of susceptible and resistant rat strains.

Varying results have been reported on the role of neu oncogene in mammary carcinogenesis. In order to further address this issue, the activated neu oncogene was introduced into mammary epithelial cells in situ of both mammary carcinoma-susceptible Wistar Furth and resistant Copenhagen rats by infusing replication-defective recombinant retroviruses carrying the neu oncogene into the mammary gland lumen. At the highest virus titer tested, very high numbers of mammary carcinomas developed within 2 weeks in all exposed glands in both rat strains. When the virus titer was reduced, however, individual tumors occurred with varying latencies. In addition, not all of the neu-infected mammary cells progressed to form mammary carcinomas. These results suggest that while neu is a potent mammary transforming gene, either other events in addition to neu expression may be required for full malignant transformation or not all mammary ductal epithelial cells are able to be neoplastically transformed.

Difference in the response of neu and ras oncogene-induced rat mammary carcinomas to early and late ovariectomy.

Rat mammary carcinomas were induced by directly inserting activated neu or ras genes into in situ rat mammary ductal cells using replication-defective retroviral vectors. neu was over 200 times more potent than ras in inducing rat mammary carcinomas. Ovariectomy 2 days postinfection dramatically reduced the occurrence of carcinomas induced by neu and extended their latency. In general, early ovariectomy had much less effect on the occurrence of carcinomas induced by ras and had no significant effect on their latency. Carcinomas induced by neu in ovariectomized rats had down-regulated estrogen receptor and progesterone receptor, while those induced by ras had only down-regulated progesterone receptor. Fully progressed mammary carcinomas in intact rats induced by both neu and ras had a similar response to ovariectomy, with an approximate regression rate of 60%. These data suggest that the activation of ras, but not neu, can replace at least some functions performed by ovarian hormones in the early phases of mammary carcinogenesis. These data also suggest a role for antiestrogen drug therapy in the prevention of neu-associated breast cancer.

Tamoxifen-induced increase in the potential doubling time of MCF-7 xenografts as determined by bromodeoxyuridine labeling and flow cytometry.

The anti-estrogen tamoxifen (TAM) is widely used in the therapy of human breast cancer. Shown to induce a G1 transition delay in vitro, the kinetic effects of TAM on breast carcinoma cells growing as tumor xenografts in nude mice have been less well characterized. In this study, we demonstrate a significant increase in the tumor potential doubling time (Tpot) and decrease in the labeling index (%LI) of estradiol (E2)-stimulated MCF-7 xenografts following TAM treatment or E2 deprivation. MCF-7 tumor pieces were transplanted s.c. into nude mice supplemented with Silastic capsules containing E2. After 2-4 weeks, animals were randomized to continued E2 treatment, E2 and TAM treatment, or E2 deprivation. At times ranging from 0 to 23 days after treatment, animals were given injections of bromodeoxyuridine and tumors excised for kinetic analysis. Using flow-cytometric techniques, the Tpot and %LI were estimated for all tumors. Seven independent experiments were performed and data pooled for statistical analysis. At the time of hormonal manipulation, E2-stimulated tumors had a volume doubling time of 5 days, a Tpot of 2.3 days, and a %LI of 23%. Continued E2 treatment resulted in only minimal changes in Tpot and %LI over the remainder of the observation period. Treatment with TAM resulted in a slowing of tumor growth (tumor doubling time, 12 days), a significant (P < 0.001) increase in Tpot to 6.6 days, and a decrease in %LI to 8% by 23 days posttreatment. E2 deprivation resulted in a cessation of tumor growth and similar changes in Tpot and %LI to 5.3 days and 10%, respectively (P < 0.001). In contrast to previous reports, these data demonstrate that TAM treatment and E2 deprivation both significantly decrease tumor cell proliferation in MCF-7 xenografts.

Toxicity and dose-response studies of 1,25-(OH)2-16-ene-23-yne vitamin D3 in transgenic mice.

The vitamin D3 analogue 1,25-(OH)2-16-ene-23-yne vitamin D3 (16,23-D3) in doses with low systemic toxicity has been demonstrated to inhibit retinoblastoma growth in transgenic mice. This study examines the dose-dependent response for inhibition of tumor growth in transgenic mice with retinoblastoma and evaluates the in vivo toxicity of 16,23-D3 in nontransgenic mice. Transgenic 8-10-week-old mice with retinoblastoma (n = 119) were randomly assigned to groups receiving 1.0, 0.75, 0.5, 0.35, 0.2, or 0.05 microg of 16,23-D3 and a vehicle alone (control) group i.p. five times a week for 5 weeks. An additional control group received no injection. Eyes were enucleated one week after the end of treatment, and tumor areas were measured. To determine the toxic dose, transgene-negative littermates received 0.5, 1.0, 1.5, 2.5, 3.5, 4.5, or 5.0 microg of 16,23-D3, and control groups received vehicle alone, 5 days a week for 5 weeks. Serum calcium levels were measured, and necropsies were performed on animals from each group. In the dose-response study, tumor growth inhibition was greatest in the group receiving 0.35 microg (55% inhibition; P = 0.0056) and was also significant in the group receiving 0.5 microg (42% inhibition; P = 0.036). The systemic toxic effects due to hypercalcemia occurred at doses of > or =1.0 microg. 16,23-D3 inhibits tumor growth at doses > or =0.35 microg and shows toxic effects at doses > or =1.0 microg related to hypercalcemia in mice fed an unrestricted diet. No toxicity was observed with lower doses.

In vivo modulation of iododeoxyuridine metabolism and incorporation into cellular DNA by 5'-amino-5'-deoxythymidine in normal mouse tissues and two human colon cancer xenografts.

This in vivo study examines the ability of 5'-amino-5'-deoxythymidine (5'-AdThd) to modulate 5-iododeoxyuridine (IdUrd) cellular metabolism in two human colon cancer xenografts (HT 29 and HCT-116), two actively proliferating normal mouse tissues (bone marrow and intestine), and a quiescent normal mouse tissue (liver). 5'-AdThd is a thymidine analogue that at low concentrations (<30 micrometer) can increase thymidine kinase activity, which is the rate-limiting enzyme for activation of IdUrd. We reported recently that the in vitro incubation of HT 29 and HCT-116 cells in 5'-AdThd + IdUrd resulted in an enhancement of 5-iodo-2'-dUTP pools, IdUrd DNA incorporation, and subsequent radiosensitization compared with incubation with IdUrd alone (Clin. Cancer Res., 1: 407-416, 1995). These in vitro effects were more significant in the radioresistant cell line HT 29. Using a 6-day continuous infusion of IdUrd (50 or 100 mg/kg/day) and/or 5'-AdThd (200 mg/kg/day), no increase in systemic toxicity (percentage of body weight loss) was observed in athymic nude mice with 5'-AdThd alone or when combined with IdUrd. There was significant dose-dependent, systemic toxicity with IdUrd, which was reversible within 3 days of completing the lower-dose IdUrd infusion. However, a comparison of plasma levels during the 6-day continuous infusion of IdUrd +/- 5'-AdThd showed a significant interaction of IdUrd and 5'-AdThd, resulting in higher plasma levels by day 6 of both compounds and the principal metabolites, iodouracil and deoxyuridine, which is consistent with nonlinear saturating effects on dihydrouracil dehydrogenase. Coadministration of IdUrd and 5'-AdThd resulted in an increase in the percentage of IdUrd DNA incorporation in the two proliferating normal tissues, which was significant only with the lower IdUrd dose. No effect on IdUrd DNA incorporation was found in normal liver at either IdUrd dose +/- 5'-AdThd. Similar to our in vitro data, the continuous infusion of IdUrd and 5'-AdThd showed a significant effect by increasing the percentage of IdUrd DNA incorporation in HT-29 xenografts at both IdUrd doses, whereas coadministration of 5'-AdThd had no such effect in HCT-116 xenografts.

Differential quantitative effects of interleukin (IL)-2 and IL-15 on cytotoxic activity and proliferation by lymphocytes from patients receiving in vivo IL-2 therapy.

Lymphocytes from patients receiving in vivo interleukin (IL)-2 therapy possess enhanced in vitro proliferative and cytotoxic responses to IL-2. The cells from these patients that respond to exogenous IL-2 are CD56+ natural killer cells expressing intermediate-affinity IL-2 receptor betagamma(c) complexes. Because IL-15 activates cells via these same betagamma(c) receptors, we hypothesized that IL-15 would also activate lymphocytes from patients treated with in vivo IL-2 therapy and therefore that IL-15 might potentially be useful as an immunotherapeutic agent alone or in combination with IL-2. We report here that peripheral blood mononuclear cells (PBMCs) from patients receiving in vivo IL-2 therapy do proliferate in response to IL-15. However, a greater dose of IL-15 is needed to reach the same level of proliferation stimulated by IL-2. The EC50 for IL-2 is 0.21 +/- 0.04 nM (mean +/- SE; n = 18), whereas the EC50 for IL-15-stimulated proliferation is 1.16 +/- 0.16 nM (n = 18). In contrast to the proliferative response, equivalent doses of IL-2 and IL-15 stimulate patient PBMCs to mediate similar levels of cytotoxicity against Daudi, K562, and LA-N-5 tumor targets. Notably, low concentrations of IL-15 that do not stimulate a substantial proliferative response (e.g., 1.0 ng/ml) do boost PBMCs to mediate cytotoxicity against these tumor targets. These distinct dose-response curves for proliferation compared to cytotoxicity suggest that IL-15 should be evaluated for its potential as an immunotherapeutic agent to treat cancer, particularly in regimens providing doses that might minimize the proliferative response (associated with cytokine release and toxic side effects) while maintaining the cytolytic antitumor response.

Leucovorin modulation of 5-iododeoxyuridine radiosensitization: a phase I study.

Evidence for clinically significant radiosensitization by the halogenated pyrimidine 5-iododeoxyuridine (IdUrd) continues to accumulate. In vitro radiosensitization has been demonstrated in human colon tumor cell lines following exposure to 1-10 micrometer. Coadministration of leucovorin (LV) increases radiosensitization, which correlates directly with increased IdUrd DNA incorporation. Clinical data regarding proliferation rates and thymidine kinase levels in tumors versus normal tissues suggest selective incorporation of IdUrd into gastrointestinal tumors may occur. The objectives of this Phase I study were: (a) to assess the feasibility of LV modulation of IdUrd radiosensitization by determining the maximum tolerated dose (MTD) of IdUrd plus LV; and (b) to perform correlative laboratory studies to investigate the potential of IdUrd plus LV to increase radiosensitization in vivo. Seventeen patients with unresectable or recurrent gastrointestinal adenocarcinomas received a 14-day course of continuous i.v. infusion of IdUrd prior to initiation of radiotherapy. Two additional 14-day infusions of IdUrd with LV were given during the course of radiotherapy (60 Gy in 6 weeks). The initial dose of IdUrd was 250 mg/m2/day and was escalated in subsequent patients to 400 and 600 mg/m2/day. The LV dose remained fixed at 250 mg/m2/day. Leukopenia was the dose-limiting toxicity, and 400 mg/m2/day was the MTD for this trial. At the MTD, the mean +/- SD steady-state plasma concentration of IdUrd during the infusion, measured by high-performance liquid chromatography, was 0.66 +/- 0.23 micrometer. There was no significant influence of LV on IdUrd DNA incorporation in peripheral blood granulocytes as measured by high-performance liquid chromatography. Based on toxicity data and correlative laboratory studies, a meaningful increase in radiosensitization would not be achieved with the IdUrd infusion schedule and dose of LV investigated compared with IdUrd alone.

2',5'-oligoadenylate synthetase, neopterin and beta 2-microglobulin in asymptomatic HIV-infected individuals.

This study examined 2',5'-oligoadenylate (2,5A) synthetase activity in 26 individuals during the asymptomatic phase of HIV infection and its correlation with neopterin or beta 2-microglobulin. In HIV-antibody-positive (HIV-Ab+) asymptomatic people, both neopterin and beta 2-microglobulin levels in sera were significantly elevated; in contrast, 2,5A-synthetase activity in peripheral blood mononuclear cells was not significantly higher than in HIV-antibody-negative controls. The 2,5A-synthetase levels in symptomatic people (AIDS-related complex and AIDS) were significantly higher than in either asymptomatic or control individuals. However, within the group of HIV-infected asymptomatic individuals, all three markers were positively correlated. In this group, neopterin values were negatively correlated with the number of CD4+ lymphocytes while a positive correlation was found between 2,5A-synthetase and the number of CD8+ lymphocytes. Asymptomatic people with detectable serum HIV p24 antigen had significantly higher 2,5A-synthetase, neopterin, beta 2-microglobulin and number of CD8+ lymphocytes. This study suggests that elevated 2,5A-synthetase activity may reflect a different aspect of host response to HIV infection than do elevated neopterin or beta 2-microglobulin.

Influence of estradiol and tamoxifen on susceptibility of human breast cancer cell lines to lysis by lymphokine-activated killer cells.

The design of combination hormonal and immunotherapeutic protocols for breast cancer patients may be facilitated by analysis of preclinical in vitro model systems. Estrogen receptor positive (ER+: MCF-7) and negative (ER-: MDA-MB-231) human breast cancer cell lines were utilized to evaluate the effects of tamoxifen (TAM) and estradiol (E2) on modulation of breast cancer target susceptibility to lysis by lymphokine-activated killer (LAK) cells. E2-stimulated ER+ cells were more susceptible to lysis by LAK cells than corresponding TAM-treated or control cells, while treatment of ER- cells with either E2 or TAM alone did not alter from control their susceptibility to this immune-mediated lysis. All ER+ and ER- cells tested remained sensitive after treatment with TAM to lysis by LAK cells. In addition, an adenocarcinoma reactive human-mouse chimeric monoclonal antibody (ING-1) was able to significantly boost in vivo generated LAK cell-mediated lysis of control, E2-treated, and TAM-treated ER+ and ER- cells. These in vitro results provide a preclinical rationale for in vivo testing of TAM, interleukin-2 (IL-2), and breast cancer reactive antibody-dependent cellular cytotoxicity facilitating antibody in patients with refractory or high risk breast cancer.

Re-analysis of the time factor in local control by radiotherapy of T3T4 squamous cell carcinoma of the larynx.

The use of logistic regression for the analysis of local control data is illustrated via a re-analysis of previously published data. A larger effect of overall time is found than in the original publication. In addition, the original analysis of 50 percent effective dose (ED50) is found to have obscured the relationship between local control and overall time for some doses.

Loss of local control with prolongation in radiotherapy.

Twelve published clinical results of radical radiotherapy of head and neck cancer have been reviewed, seven of them with fresh multivariate analyses, to determine the magnitude of time factors relating local control to overall time. In all but two of the data sets a significant loss of local control was observed with prolongation. The median rate of loss was 14% in only 1 week, the range 3 to 25%. This corresponds to a median loss of 26% in 2 weeks (5-42%). These results are comparable with other, less detailed information. Whether these significant losses are due to proliferation of tumor cells or to other causes such as physician selection, it is clear that modest prolongation is associated with a lower chance of local control.

The decreased influence of overall treatment time on the response of human breast tumor xenografts following prolongation of the potential doubling time (Tpot).

PURPOSE: Repopulation during fractionated radiotherapy has been postulated to result in a significant loss in local control in rapidly proliferating tumors. Clinical data suggest that accelerated fractionation schedules can overcome the influence of repopulation by limiting the overall treatment time. Unfortunately, accelerated therapy frequently leads to increased acute reactions, which may become dose limiting. An alternative to accelerated fractionation would be to decrease the rate of repopulation during therapy. To test the potential efficacy of this alternative, we examined the effect of reducing tumor proliferation rate on the response of MCF-7 human breast carcinoma xenografts treated with a short vs. a long course of fractionated therapy. To reduce the proliferation rate, we deprived nude mice transplanted with MCF-7 xenografts of the growth-stimulating hormone estradiol (E2). We have previously reported that E2 deprivation increases the potential doubling time (Tpot) for MCF-7 xenografts from a mean of 2.6 days to 5.3 days (p < 0.001). METHODS AND MATERIALS: E2-stimulated and E2-deprived MCF-7 breast carcinoma xenografts were clamped hypoxically and irradiated with four fractions of 5 Gy each, using either a short (3-day) or long (9-day) treatment course. E2 stimulation was restored in all animals at the completion of irradiation. Radiation response was determined by regrowth time and regrowth delay of the irradiated tumors as compared to unirradiated controls. RESULTS: Prolongation of therapy in rapidly proliferating, E2-stimulated tumors (Tpot approximately 2.6 days) resulted in a significant decrease in regrowth time in two identical experiments. With results pooled for analysis, the regrowth times for the short and long treatments were 62 and 32 days, respectively (combined p < 0.001). The shorter regrowth times suggest that there was less overall tumor damage with the longer fractionated radiotherapy course. No significant difference in regrowth time was observed in the more slowly proliferating, E2-deprived tumors (Tpot approximately 5.3 days) treated with either the short or long regimen. Median regrowth times were 48 and 54.5 days for the short and long treatments, respectively (combined p = 0.14). Similar changes were observed in regrowth delay. CONCLUSIONS: Reduction in the rate of cell proliferation, induced by E2 deprivation in MCF-7 human breast xenografts during fractionated radiotherapy, resulted in a significantly decreased dependence on overall treatment time in comparison to the more rapidly proliferating E2-stimulated tumors. This model suggests that pharmacologically induced reduction in the rate of tumor cell proliferation during a course of fractionated radiotherapy may be a viable alternative to accelerated fractionation for the treatment of rapidly proliferating tumors.

Single biopsy, tumor kinetic analyses: a comparison of methods and an extension to shorter sampling intervals.

There is emerging and established clinical and laboratory evidence that proliferation of tumor clonogens during radiation therapy can impair local tumor control. The pre-treatment, tumor potential doubling time, T(pot), estimated with in situ bromodeoxyuridine (BrdUrd) labeling, followed by a single biopsy and flow cytometry, may be a predictor of a given tumor's ability to undergo such intra-treatment proliferation. Recent studies have found a strong similarity between T(pot)'s determined in this fashion and the effective doubling times of surviving tumor cells during radiotherapy, as estimated from tumor control versus treatment duration data. Furthermore, several preliminary clinical studies have indicated that T(pot) may be a predictor of outcome, with faster tumors doing worse. Accelerated fractionation might overcome such proliferation, but is more acutely toxic and is unlikely to benefit patients with slowly proliferating tumors. Thus, the BrdUrd/single biopsy method may offer the possibility of selecting between accelerated and conventional or hyperfractionated treatment. Several approaches to the analysis of data generated by this method have been described, but there has been little documentation of the validity of methods in experimental systems, particularly in human experimental tumors. This study explores various analytic methods employed with the BrdUrd, delayed, single-biopsy technique used in determining the potential doubling time, T(pot), of tumors. It compares methods of analysis in three experimental systems and in 40 in situ-labeled human tumors, and proposes a method for shortening the required labeling-biopsy interval to a clinically more convenient range of 3 to 4 hours.

The role of cell cycle redistribution in radiosensitization: implications regarding the mechanism of fluorodeoxyuridine radiosensitization.

PURPOSE: Radiosensitization has previously been demonstrated in a human colon cancer cell line (HT-29) following a 2 h exposure to low, clinically relevant concentrations (0.05-0.5 microM) of fluorodeoxyuridine (FdUrd) (15). The sensitizer enhancement ratio value (measured at 10% survival) plateaued at approximately 1.7 between 16 and 32 h following removal of drug. Parallel studies investigating the effect of FdUrd on the distribution of cells throughout the cell cycle found that the percentage of cells in early S-phase increased to approximately 70% during the same period that maximal radiosensitization was noted. As a follow-up to these findings, experiments have been designed to investigate the contribution of this early S-phase delay to radiosensitization. METHODS AND MATERIALS: Synchronized populations of HT-29 cells have been obtained with three separate techniques. Two involve the induction of a reversible metaphase arrest (with high pressure N2O or colcemid) followed by a shakeoff of mitotic cells. The third uses a plant amino acid, mimosine, to induce a reversible block at the G1/S boundary. Flow cytometry was used to analyze the degree of synchrony based on bromodeoxyuridine (BrdUrd) uptake and propidium iodide (PI) staining. Radiation survival curves were obtained on these synchronized populations to investigate changes in radiosensitivity through the cell cycle. Additionally, levels of thymidylate synthase (TS), the primary target of FdUrd cytotoxicity, were measured in each phase of the cell cycle using the TS 106 monoclonal antibody against human TS. RESULTS: Synchronization with mitotic shakeoff produced relatively pure populations of cells in G1; however, the degree of synchrony in early S-phase was limited both by cells remaining in G1 and by cells progressing into late S-phase. These techniques failed to reveal increased radiosensitivity in early S-phase at 10% survival. An 18 h exposure to mimosine resulted in populations that more closely resembled the early S-phase enrichment following FdUrd exposure and revealed increased radiosensitivity during early S-phase. TS levels were noted to be only 1.3 times higher in S phase than in G0/G1. CONCLUSION: Radiation survival data from cells synchronized with mitotic shakeoff techniques suggest that early S-phase delay is unlikely to be the primary mechanism of FdUrd radiosensitization. In contrast, the increased sensitivity seen in early S-phase with mimosine synchronized cells is similar to that seen with FdUrd. Although confounding biochemical pertubations cannot be ruled out, these data continue to suggest an association between early S-phase enrichment and radiosensitization. The significance of TS inhibition as a mechanism of FdUrd radiosensitization remains unclear.