A phase II trial of weekly intravenous gemcitabine and cisplatin with continuous infusion fluorouracil in patients with metastatic renal cell carcinoma.

BACKGROUND: We reported previously that the combination of gemcitabine and continuous infusion fluorouracil (5-FU) has activity in renal cell carcinoma. Based upon in vitro synergy of gemcitabine/cisplatin and 5-FU/cisplatin, we hypothesized that the addition of cisplatin could improve the objective response rate of gemcitabine and 5-FU with manageable toxicity. PATIENTS AND METHODS: Twenty-one patients with metastatic renal cell carcinoma (RCC) and a Cancer and Leukemia Group B performance status of 0 to 2 were enrolled. Ten had received prior systemic therapy. Treatment consisted of gemcitabine 600 mg/m2 and cisplatin 20 mg/m2 on days 1, 8 and 15 of each 28-day cycle. Continuous infusion 5-FU was given from day 1 to day 21. RESULTS: No complete responses and one partial response were observed for an objective response rate of 5% (95% confidence interval 0% to 24%). Two minor responses (25% to 50% regression) were also observed. The median overall survival was 10 months with 35% of patients surviving at 1 year. Grade 3-4 myelosuppression (mostly thrombocytopenia) occurred in nine (43%) patients. Nausea/vomiting and neuropathy were dose-limiting in an additional five patients. Only 51% of treatment cycles were delivered on time and without dose reduction. CONCLUSIONS: The addition of cisplatin to gemcitabine and 5-FU did not improve the objective response rate of gemcitabine and 5-FU alone and added to the toxicity. Due to the cumulative toxicity, further trials with this cisplatin-containing regimen in RCC are not indicated.

Sequence-dependent antagonism between fluorouracil and paclitaxel in human breast cancer cells.

The effects of 24-hr exposures to 5-fluorouracil (FUra) and paclitaxel in various sequences were studied in MCF-7 breast cancer cells to determine an optimal schedule for possible clinical use. In clonogenic assays, pre-exposure to FUra followed by paclitaxel resulted in marked antagonism, while sequential paclitaxel followed by FUra was optimal. Concurrent or pre-exposure to paclitaxel did not affect [3H]FUra metabolism, [3H]FUra-RNA incorporation, or the extent of FUra-mediated thymidylate synthase inhibition. Paclitaxel led to G2/M phase accumulation that persisted for up to 24 hr after drug exposure, while a 24-hr FUra exposure produced S-phase accumulation. FUra pre-exposure diminished paclitaxel-associated G2/M phase block, whereas subsequent exposure to FUra after paclitaxel did not. FUra exposure resulted in transient induction of p53 and p21, which returned to basal levels 24 hr after drug removal. p53 and p21 protein content also increased markedly during paclitaxel exposure, accompanied by phosphorylation of Bcl-2. Double-stranded DNA fragmentation (approximately 50 kb) was seen at 48 hr when cells were exposed to paclitaxel for an initial 24-hr period. Paclitaxel-associated DNA fragmentation was not prevented by concurrent or subsequent exposure to FUra. Thus, paclitaxel-mediated G2/M phase arrest appeared to be a crucial step in induction of DNA fragmentation. Since an initial 24-hr paclitaxel exposure did not interfere with subsequent FUra metabolism or thymidylate synthase inhibition, and delayed exposure to FUra did not impede either paclitaxel-mediated induction of mitotic blockade or DNA fragmentation, the sequence of paclitaxel followed by FUra is recommended for clinical trials.

Relative frequencies and sites of presentation of lymphoid neoplasms in a community hospital according to the revised European-American classification.

Relative frequencies for common subtypes in the revised European-American classification of lymphoid neoplasms (REAL classification) have been reported. We determined the relative frequencies and sites of presentation of REAL subtypes at a 700-bed community hospital in central Illinois. A database was used to identify and prospectively catalogue all newly diagnosed lymphoid neoplasms from July 1, 1995 to March 1, 1998. The approach to diagnosis and subtyping incorporated morphologic features, immunophenotype, and clinical findings according to criteria proposed in the REAL classification. Of 347 lymphoid neoplasms diagnosed, 319 were subtyped in the REAL classification. Of these, 261 were B-cell neoplasms, 21 were T-cell neoplasms, and 37 were Hodgkin disease variants. Chronic lymphocytic leukemia/small lymphocytic lymphoma/prolymphocytic leukemia, diffuse large cell, and follicle center neoplasms were the most common B-cell subtypes. Large granular lymphocyte leukemia was the most common T-cell neoplasm. Nodular sclerosis was the most common Hodgkin disease variant. The relative frequencies in a US community hospital setting are similar to those reported in other studies. Differences are attributable to patient selection criteria, study group geographic location and racial composition, and/or referral patterns. Diverse REAL classification subtypes may be expected in US community hospitals.

Modulation of fluorouracil cytotoxicity by interferon-alpha and -gamma.

Because interferons (IFN)-alpha and -gamma individually have increased fluorouracil (FUra) cytotoxicity in several in vitro models, we studied the effects of FUra combined with IFN-alpha + gamma in HT29 colon cancer cells. A 96-hr exposure to IFN-alpha (500 units/ml) plus IFN-gamma (10 units/ml) and a 72-hr exposure to 0. 25-1 microM FUra (hr 24-96) inhibited cell growth and colony formation in an additive or more-than-additive fashion. When cells were exposed to IFN-alpha + gamma and FUra, free FdUMP levels became detectable, whereas [3H]FUra-RNA incorporation decreased. Exposure to IFN-alpha + gamma, FUra, or the combination decreased dTTP pools to 58%, 43%, and 17% of control, respectively. A marked increase in the dATP to dTTP ratio was seen with FUra with or without IFN-alpha + gamma. Thymidylate synthase catalytic activity was reduced to 28% and 24% of control with FUra with or without IFN-alpha + gamma, suggesting that the enhanced dTTP depletion must be due to another mechanism. FUra-mediated thymidylate synthase inhibition was accompanied by a 124-fold increase in total deoxyuridylate immunoreactivity and a 31-fold increase in dUTP pools, but the addition of IFN-alpha + gamma attenuated the accumulation. Treatment with IFN-alpha + gamma and FUra individually interfered with nascent DNA chain elongation, whereas the three-drug combination produced the most striking effects. IFN-alpha + gamma plus FUra produced the greatest amount of single-strand breaks in nascent DNA and dramatically decreased net DNA synthesis. IFN-alpha + gamma with or without FUra produced double-strand breaks in parental DNA. These results suggest that dTTP depletion, dATP/dTTP imbalance, pronounced inhibition of DNA synthesis, and damage to nascent and parental DNA contribute to the enhanced cytotoxicity with the triple combination.

Determinants of cytotoxicity with prolonged exposure to fluorouracil in human colon cancer cells.

To explore the determinants of cytotoxicity during prolonged exposure to pharmacologically relevant concentrations of 5-fluorouracil (FUra), we studied the effects of FUra at concentrations ranging from 0.1 to 1 microM in HCT 116 and HT 29 colon cancer cells grown in the presence of physiologic levels of leucovorin. A 5- and 7-day exposure to 1 microM FUra reduced cell growth to 46% and 20% of control in HT 29 cells and to 74% and 38% of control in HCT 116 cells. Concurrent exposure to thymidine (10 or 20 microM) or uridine (1 mM) provided partial protection against FUra toxicity in HT 29 cells, but did not protect HCT 116 cells. After a 24-h exposure to 1 microM [3H]FUra, free 5-fluoro-2'-deoxyuridine-5' -monophosphate (FdUMP) and FUDP. + FUTP levels were 0.7 and 144 pmol/10(6) cells in HT 29 cells, respectively, and 3.9 and 178 pmol/10(6) cells in HCT 116 cells. FdUMP and FUDP + FUTP pools increased by 5.7- and 2.0-fold in HT 29 cells and by 1.7- and 3.3-fold in HCT 116 cells over the next 48 h, but did not accumulate thereafter. After a 24-h exposure to 1 microM [3H]FUra, FUra-RNA levels were 158 and 280 fmol/microgram in HT 29 and HCT 116 cells, respectively; FUra-RNA levels increased over time, and reached 700 and 1156 fmol/microgram at day 5. Concurrent exposure to 1 mM uridine for 72 h did not diminish [3H]FUra-RNA incorporation. Upon removal of [3H]FUra following a 24-h exposure, FUra-RNA levels remained relatively stable with 57-78% retained at 120 h. A low level of [3H]FUra-DNA incorporation was detected in HT 29 cells. Thymidylate synthase (TS) catalytic activity in control cells was 2-fold higher in HCT 116 cells compared to HT 29 cells (47 vs. 23 pmol/min/mg). Total TS content increased 1.5- to 3-fold over control in both cell lines during FUra exposure, and ternary complex formation was evident for up to 96 h-dTTP pools were not depleted in FUra-treated cells, suggesting that residual TS catalytic activity was sufficient to maintain dTTP pools relative to demand. Surprisingly, the partial inhibition of TS was accompanied by a striking accumulation of immunoreactive "dUMP" pools in both lines; dUTP pools also increased 2-to 3-fold. In summary, the gradual and stable accumulation of FUra in RNA noted in both lines may account for the thymidine-insensitive component of FUra toxicity. Because dTTP pools were not appreciably diminished, the interference with nascent DNA chain elongation and induction of single-strand breaks in newly synthesized DNA in both cell lines may be due to misincorporation of deoxyuridine nucleotides.

High affinity human antibodies by phage display.

The development of phage display has now made it possible to consider the isolation of human antibodies directly without immunization. Recent advances in the field of human immunogenetics and in phage technology have led to the assembly of 'naive' human repertoires in vitro whose complexity approach that of the natural immune system. Screening of these libraries has allowed the isolation in one step of antibodies with affinities in the nanomolar range.

Full-scale 'naïve' human antibody repertoires assembled from VH and VL variable regions.

Very large 'naïve' human antibody repertoires have been obtained from RT-PCR cloned VH and VL variable regions. They are used as starting material for the assembly of medium sized combinatorial libraries or so called multicombinatorial libraries. In nonimmunized individuals immunoglobulin messenger RNAs are poorly expressed, which can be a serious limitation for cloning efficiency. To overcome this problem two complementary strategies have been used: a nonspecific polyclonal activation of B cells, and a secondary PCR amplification technique to ensure recovery of Ig messengers in large amount and without introducing any bias.

Murine cytomegalovirus inactivated by sodium periodate is innocuous and immunogenic in mice and protects them against death and infection.

Sodium periodate (10 mM, 4 degrees C) inactivated murine cytomegalovirus (MCMV) very rapidly (loss of 2 to 3 log of viral infectivity per minute). Periodate-treated MCMV (PI-MCMV) was shown to be innocuous in mice, as determined by the inability of the virus to replicate. PI-MCMV induced a strong humoral immune response, with a high level of neutralizing antibodies. Mice immunized with PI-MCMV were protected against death and infection, when a lethal challenge with the virulent virus was administered 3 weeks after immunization and from death but not infection when virulent virus was administered at 3 months. Finally, no reactivation of potentially latent challenge virus (sublethal dose at 3 weeks) was observed in animals immunosuppressed at 6 months after immunization. Taken together, these results suggest that periodate could serve as an inactivating agent to prepare killed vaccines.

Effects of 9-aminocamptothecin on newly synthesized DNA in patient bone marrow samples.

9-Aminocamptothecin (9-AC) inhibited cell growth and DNA synthesis in HCT 116 human colon cancer cells in a concentration- and time-dependent manner. Interference with nascent DNA chain elongation was monitored using pH step alkaline elution. After a 3-day 9-AC exposure, 38% (10 nM) and 53% (50 nM) of the total [3H]DNA eluted with pH steps 11.3-11.7, compared to 9% in control cells. Effects on nascent DNA integrity were also evaluated by fixed elution with pH 12.1 buffer. After a 3-day exposure to 9-AC, 27% (10 nM) and 82.5% (50 nM) of the total [3H]DNA eluted relative to control. Paired bone marrow samples were then obtained in 10 patients before treatment and between 42 and 72 h of a continuous i. v. infusion of 9-AC (35-74 microgram/m2/h for 72 h). The mononuclear cells were incubated with [3H]dThd for 2 or 4 h, and then analyzed using either pH step or fixed pH alkaline elution, respectively. In seven patients receiving >/=47 microgram/m2/h 9-AC, 4% +/- 1.5% (mean +/- SE) of the total [3H]DNA eluted with pH steps /=59 microgram/m2/h 9-AC (n = 7). Since hematological toxicity is dose limiting on this 9-AC schedule, these cellular pharmacodynamic studies provide evidence of a DNA-directed cytotoxic effect of 9-AC in a sensitive host target tissue.

Determinants of sensitivity to 1-beta-D-arabinofuranosylcytosine in HCT 116 and NCI-H630 human colon carcinoma cells.

The cytotoxicity and metabolism of 1-beta-D-arabinofuranosylcytosine (AraC) and its effects on DNA synthesis and integrity were studied in HCT 116 and NCI-H630 human colon cancer cells. In 116 cells, 0.1 microM AraC decreased colony formation by approximately 50%, whereas 1 microM was required in H630 cells. AraCTP levels after a 24-hr AraC exposure were 2.3- to 3.5-fold lower in H630 cells due to increased ability to deaminate AraCMP. AraC DNA levels increased in proportion to AraCTP pools (r = 0.99) and were 2-fold higher in 116 cells after a 24-hr exposure to 0.1 and 1 microM AraC. Although the half-life of AraCTP was < 1 hr in both lines, > 80% of AraC DNA was retained at 24 hr after drug removal. Clonogenic capacity was inversely related to the extent of AraC DNA incorporation. Interference with nascent DNA chain elongation increased with increasing AraC concentration x time. A 24-hr AraC exposure produced a dramatic shift in the elution profile of nascent DNA during a 15-hr elution at pH 12.1; these effects were greater in 116 cells (DNA retained on filter [percentage of control]): 78%, 23%, and 9% with 0.1, 1, and 10 microM AraC versus 84%, 42%, and 18% in H630 cells, respectively. The extent of nascent DNA damage was proportional to AraC DNA content. Net DNA synthesis was potently inhibited during AraC exposure in both lines. H630 cells had partial recovery of DNA synthesis at 24 hr after drug removal, whereas persistent inhibition was noted in 116 cells. A slight excess of double-strand breaks in parental DNA was detected after a 24-hr exposure to 10 microM AraC in 116 cells. The extent of DNA fragmentation was more pronounced 16 hr after drug removal and was greater in 116 cells: 8.5%, 19%, and 21% with 0.1, 1, and 10 microM AraC DNA content, magnitude of nascent DNA damage, duration of DNA synthetic inhibition, and induction of double-stranded DNA fragmentation appeared to be the crucial determinants of lethality.

A new phage display system to construct multicombinatorial libraries of very large antibody repertoires.

We present an easy and efficient technique for the construction of large phage-displayed antibody (Ab) repertoires through the recombination of two separate heavy (VH) and light (VL) chain gene libraries. Here, the system has been applied to the display of a chimpanzee anti-HIV gp160 Ab. The process, which makes use of lambda phage att recombination sites, leads to the irreversible physical association between plasmid and phagemid vectors carrying, respectively, VL and VH sequences. The heat-inducible expression of the Int recombinase allows perfect control of recombination. Selection of the recombinant phagemid is made possible by the assembly, in vivo, of a genetic marker (chloramphenicol resistance) created only after the correct recombination event. Theoretically, all possible associations between the VL and VH sequences should be obtained, and it should be possible to generate multicombinatorial libraries of close to 10(12) clones.

Determinants of trimetrexate lethality in human colon cancer cells.

We examined the cytotoxicity and biochemical effects of the lipophilic antifol trimetrexate (TMQ) in two human colon carcinoma cell lines, SNU-C4 and NCI-H630, with different inherent sensitivity to TMQ. While a 24 h exposure to 0.1 microM TMQ inhibited cell growth by 50-60% in both cell lines, it did not reduce clonogenic survival. A 24 h exposure to 1 and 10 microM TMQ produced 42% and 50% lethality in C4 cells, but did not affect H630 cells. Dihydrofolate reductase (DHFR) and thymidylate synthase were quantitatively and qualitatively similar in both lines. During drug exposure, DHFR catalytic activity was inhibited by > or = 85% in both cell lines; in addition, the reduction in apparent free DHFR binding capacity (< or = 20% of control), depletion of dTTP, ATP and GTP pools and inhibition of [6-3H]deoxyuridine incorporation into DNA were similar in C4 and H630 cells. TMQ produced a more striking alteration of the pH step alkaline elution profile of newly synthesised DNA in C4 cells compared with 630 cells, however, indicating greater interference with DNA chain elongation or more extensive DNA damage. When TMQ was removed after a 24 h exposure to 0.1 microM, recovery of DHFR catalytic activity and apparent free DHFR binding sites was evident over the next 24-48 h in both cell lines. With 1 and 10 microM, however, persistent inhibition of DHFR was evident in C4 cells, whereas DHFR recovered in H630 cells. These data suggest that, although DHFR inhibition during TMQ exposure produced growth inhibition, DHFR catalytic activity 48 h after drug removal was a more accurate predictor of lethality in these two cell lines. Several factors appeared to influence the duration of DHFR inhibition after drug removal, including initial TMQ concentration, declining cytosolic TMQ levels after drug removal, the ability to acutely increase total DHFR content and the extent of TMQ-mediated DNA damage. The greater sensitivity of C4 cells to TMQ-associated lethality may be attributed to the greater extent of TMQ-mediated DNA damage and more prolonged duration of DHFR inhibition after drug exposure.

Cytotoxicity and metabolism of 4-methoxy-8-(beta-D-ribofuranosylamino)pyrimido[5,4-d]pyrimidine in HCT 116 colon cancer cells.

We examined the cytotoxicity, biochemical effects and metabolism of 4-methoxy-8-(beta-D-ribofuranosylamino)pyrimido[5,4-d]pyrimidine (MRPP), a synthetic nucleoside inhibitor of phosphoribosylpyrophosphate synthetase, in HCT 116 human colorectal cancer cells. A 4-hr exposure to 1 and 10 microM MRPP inhibited cell growth over a 72-hr period by 76 and 89%, and inhibited clonogenic capacity by 36 and 65%, respectively. MRPP was avidly metabolized to the 5'-monophosphate derivative (MRPP-MP), and MRPP-MP formation increased with increasing MRPP exposure (microM.hr). MRPP-MP was stable, and the intracellular half-life was in excess of 48 hr. A 4-hr exposure to 10 microM MRPP resulted in significant decreases in ATP, UTP, GTP, CTP, dATP, dTTP, and PRPP pools. Near maximal ribonucleotide triphosphate depletion was achieved with > or = 24 microM.hr MRPP, and growth inhibition as a function of MRPP microM.hr closely reflected the biochemical effects. Ribonucleotide triphosphate pools remained depleted for up to 48 hr after drug removal, apparently as a consequence of the prolonged retention of MRPP-MP. MRPP (10 microM) inhibited the salvage of [3H]guanine, [3H]adenine and [3H]guanosine, and concurrent exposure to MRPP and either 100 microM adenine, hypoxanthine, or guanine did not reverse ATP or GTP depletion. Concurrent exposure to 10 microM MRPP and either 10 microM adenosine, uridine or thymidine was accompanied by repletion of ATP, UTP, and dTTP pools, respectively, but depletion of other nucleotide pools was not corrected. In contrast, 10 microM guanosine did not correct GTP depletion in the presence of MRPP. The combination of 10 microM each of thymidine, uridine, adenosine and guanosine during and following a 24-hr exposure to MRPP provided partial protection against 0.1 or 1 microM MRPP, but did not affect the cytotoxicity associated with 10 microM MRPP. MRPP is a novel antimetabolite that inhibits both de novo and salvage pathways for purine synthesis and de novo pyrimidine synthesis.

Chemotherapy of advanced gastrointestinal cancer.

Pancreatic, gastric, and colorectal carcinomas are diagnosed in 200,000 Americans each year. Therapeutic options for patients with advanced disease are limited; conventional chemotherapy is palliative and produces complete responses in only a few patients. Clinical research has focused on the evaluation of investigational new drugs, combination regimens, and biochemical modulation of fluorouracil. Unfortunately, the results of recent phase II studies of new agents have been disappointing. The exception is CPT-11, a topoisomerase I inhibitor, which showed promising activity in colorectal cancer (in including patients who had failed prior fluorouracil therapy). Modified regimens of fluorouracil and methotrexate with either doxorubicin alone or with epirubicin and cisplatin were associated with response rates approaching 50% in patients with gastric cancer, but appeared to be less toxic than previously published regimens. A randomized trial comparing fluororacil alone or with oral leucovorin allowed early dose escalation according to individual tolerance; the response rate to fluorouracil alone (23%) was higher than that reported in previous phase III trials, suggesting the importance of using adequate doses to produce toxicity in terms of clinical response. Hepatic arterial infusion of floxuridine was associated with a 39% response rate in colorectal cancer patients with disease confined to the liver for whom systemic fluorouracil therapy had failed, suggesting this approach is a reasonable therapeutic option in carefully selected patients.

Enhanced cytotoxicity with interleukin-1 alpha and 5-fluorouracil in HCT116 colon cancer cells.

Recombinant human interleukin-1 alpha (rIL-1 alpha), at concentrations that were not growth-inhibitory when given alone (100-10,000 U/ml), enhanced the growth inhibition resulting from a 72-h fluorouracil (FUra) exposure in HCT116 colon cancer cells. Median-effect analysis of clonogenic assays indicated that rIL-1 alpha, given 24 h prior to and following a 24-h exposure to FUra, increased lethality in a more than additive fashion. rIL-1 alpha did not appear to significantly affect [3H]-FUra metabolism, total [3H]-FUra-RNA incorporation or RNA retention after drug removal, inhibition of thymidylate synthase, or thymidine triphosphate pool depletion. During continuous exposure to rIL-1 alpha, transient stimulation of RNA and DNA synthesis was observed at 72 h, with a return to normal by 96 h. A 24-h exposure to 10 microM FUra altered the elution profile of newly synthesized DNA as monitored by pH step alkaline elution. An accumulation of lower-MW single-stranded DNA species was noted with FUra compared to control, accompanied by a significantly decreased proportion of DNA retained on the polycarbonate filter: 10% retained vs. 32% for control (P = 0.01). A 48-h exposure to rIL-1 alpha alone did not affect the elution profile of nascent DNA species, nor did it enhance the effects of FUra. Although FUra did not appreciably affect pulse [3H]-uridine incorporation into RNA for the initial 8-24 h of FUra exposure, progressive inhibition of net RNA synthesis was observed thereafter. FUra prevented the stimulatory effect of rIL-1 alpha on RNA synthesis, and net RNA synthesis was significantly inhibited (by 64-79% after 72 and 96 h) with the combination compared to rIL-1 alpha alone. Continuous exposure to 10 microM thymidine did not rescue cells from the lethality of FUra alone or the combination of FUra plus rIL-1 alpha, suggesting that depletion of deoxythymidine triphosphate as a consequence of thymidylate synthase inhibition was not the most important component of FUra toxicity. In contrast, 1 mM uridine provided partial protection against the toxicity of FUra alone or with rIL-1 alpha. Although uridine did not affect FUra metabolism, it decreased FUra-RNA incorporation by 42-60%, presumably as a consequence of the 2-fold expansion of UTP pools. [125I]-rIL-1 alpha binding was nonspecific; with a 24-h exposure, however, internalized [125I]-rIL-1 alpha exceeded cell surface-bound material by 2-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Inactivation of human cytomegalovirus by sodium periodate oxidation.

Human cytomegalovirus (HCMV), oxidized by sodium periodate (NaIO4), is incapable of giving rise to viral progeny in cell culture. At a NaIO4 concentration as low as 5 mM, there is a loss of at least 6 logs of viral infectivity which occurs very rapidly (less than 5 min). Further, the inactivation is a first-order reaction depending on the periodate concentration. Adsorption to the cell surface, penetration into cells, and penetration of the viral DNA into cell nuclei were found to occur identically in mock oxidized and oxidized HCMV. Since the carbohydrate moiety of viral glycoproteins was the target of periodate attack, these observations strongly suggest that the structural integrity of the sugar residues is not a prerequisite for adsorption and penetration. Nevertheless, no evidence for viral DNA or protein synthesis was detected in cells infected with oxidized virus, and even after 3 weeks in culture, no cytopathic effect was observed.