Effect of methotrexate on incorporation and excision of 5-fluorouracil residues in human breast carcinoma DNA.

We have demonstrated recently that 5-fluorouracil (FUra) residues incorporate in eukaryotic DNA. In the present study, we extend these findings and monitor the effect of methotrexate on the formation of FUra DNA. The results demonstrate that methotrexate treatment has little effect on the incorporation of FUra or 5-fluorodeoxyuridine in DNA obtained from MCF-7 human breast carcinoma cells. More importantly, we demonstrate that FUra residues are excised from MCF-7 DNA and that methotrexate enhances the excision process. This excision of FUra from eukaryotic DNA may contribute to the cytotoxicity and mutagenicity of these fluorinated pyrimidines.

Relationship between incorporation of 9-beta-D-arabinofuranosyladenine in L 1210 DNA and cytotoxicity.

We have used cesium sulfate density gradient centrifugation to monitor the incorporation of 9-beta-D-arabinofuranosyladenine (ara-A) into L1210 cellular nucleic acids. The results demonstrate the specific incorporation of ara-A in L1210 DNA. We have also found a highly significant relationship between the formation of ara-A incorporated into DNA and loss of clonogenic survival. This relationship was maintained when using ara-A in the presence of the adenosine deaminase inhibitor deoxycoformycin. Furthermore, treatment with increasing concentrations of ara-A resulted in a greater proportion of ara-A residues at the 3'-terminus, consistent with this agent providing a poor primer terminus for elongating DNA strands. These findings are similar to those obtained previously with 1-beta-D-arabinofuranosylcytosine and suggest that the incorporation of arabinofuranosyl derivatives in DNA is one mechanism responsible for cell lethality.

5-Fluorouracil incorporation in DNA of human breast carcinoma cells.

We have demonstrated previously the presence of 5-fluorouracil (FUra) residues in L1210 DNA. These findings have been extended to the MCF-7 human breast carcinoma cell line. Cesium sulfate gradient centrifugation has been used to separate the MCF-7 RNA and DNA fractions. Alkali and RNase digests have also been used to remove any possible RNA contaminating the DNA fraction. The purified DNA has been analyzed by high-pressure liquid chromatography following digestion to nucleotides and nucleosides. The results demonstrate that FUra residues are detectable in the DNA of these human breast carcinoma cells following exposure to either FUra of 5-fluorodeoxyuridine. Further, the extend of FUra incorporation in both MCF-7 RNA and DNA is similar with either fluorinated pyrimidine. We also demonstrate that the FUra incorporation in DNA from this human cell line can be enhanced by concurrent incubation with thymidine.

Breast tumor radioimmunodetection with a 111In-labeled monoclonal antibody (MA5) against a mucin-like antigen.

Monoclonal antibody MA5 recognizes a determinant displayed on high molecular weight antigens associated with secretory and malignant breast epithelial cells. MA5 reactivity with greater than 95% of primary and metastatic breast tumors, surface expression of the antigen, as well as its ability to localize within breast tumor xenografts prompted this initial study to determine the efficacy of MA5 to localize breast tumors by radioimmunoscanning. A total of 17 patients was monitored, each receiving 2 mg of purified MA5 labeled with 5 mCi of 111In. Some patients also received 3 or 18 mg of unlabeled carrier antibody (MA5); no serious allergic reactions were noted. Primary tumors, bone lesions, soft tissue recurrences, and lung metastases greater than 3 cm in diameter were detectable, whereas only one lesion (hilar node) less than 3 cm was localized. Significant antibody accumulation was noted in the liver and less significant uptake in the spleen and bone. The extensive fibrosis and poor vascularization of breast tumors may partly explain the limited sensitivity obtained thus far. The imaging results obtained with MA5 are compared with other antibodies which we show recognize the same antigens.

Patterns of antigen distribution in human carcinomas.

Ten epithelial-specific monoclonal antibodies, including monoclonal antibodies to antigens that have been used extensively in immunodiagnosis and immunotherapy experiments, were tested for reactivity with 20 human carcinomas each of the colon, lung, and breast. The antibodies tested included B72.3, OC125, and antibodies to carcinoembryonic antigen, the 17-1A antigen, and the milk fat globule mucin antigen (epithelial membrane antigen). Striking differences in the pattern of antigen distribution were seen, with each antibody having a fairly consistent staining pattern, which was dependent on the tumor type. Two antibodies reacted with most or all tumor specimens and, when positive, reacted homogeneously with apparently every cell in the specimen. Other antibodies consistently produced a variegated staining pattern, typically with areas of positive cells surrounded by areas of negative tumor cells. A third pattern was strong localization to the luminal edge and/or secretions of glandular tumors; this pattern was seen primarily in colon carcinomas which have more well-developed glandular structures than breast or lung carcinomas. A correlation with biochemical properties of the antigens was evident, in that mucins or mucin-related antigens generally produced variegated staining of lung and breast carcinomas and luminal edge/secretion staining of colon carcinomas. Such differences in antigen distribution are likely to be a major factor in developing methods for immunodiagnosis and immunotherapy.

Zoledronic acid in the treatment of hypercalcemia of malignancy: results of the international clinical development program.

This report summarizes results of the clinical development program evaluating zoledronic acid (Zometa; Novartis Pharmaceuticals Corp, East Hanover, NJ) in the treatment of hypercalcemia of malignancy (HCM). In addition to a phase I dose escalation trial, two randomized, double-blind, double-dummy studies were conducted in parallel to investigate the clinical efficacy and safety of 4 mg and 8 mg zoledronic acid in patients with moderate to severe HCM. Patients were treated with a single dose of zoledronic acid (4 or 8 mg) via 5-minute infusion or a control treatment, 90 mg pamidronate via 2-hour infusion. Patients who relapsed or had refractory HCM after initial treatment could be re-treated with 8 mg zoledronic acid. End points included rate of complete response, defined as normalization of corrected serum calcium by day 10, change in corrected serum calcium, time to relapse, duration of response, and bone biochemical markers. Doses of > or =0.02 mg/kg were effective and nontoxic in the phase I study. In the controlled studies, 287 patients were randomized and evaluated for safety and 275 patients were evaluable for efficacy. The proportions of patients with a complete response by day 10 were 88.4% and 86.7% in the 4 mg and 8 mg zoledronic acid groups, respectively, compared with 69.7% in the 90 mg pamidronate group. Corrected serum calcium normalization occurred by day 4 in 45.3% of patients treated with 4 mg zoledronic acid, 55.6% of patients treated with 8 mg zoledronic acid, and 33.3% of patients treated with pamidronate. Mean change from baseline in corrected serum calcium also was greater with zoledronic acid than with pamidronate. Median times to relapse were significantly longer in both the zoledronic acid 4 mg and 8 mg groups compared with the pamidronate group. There were no significant differences in efficacy between the 4 mg and 8 mg zoledronic acid doses. Retreatment in 69 patients with relapsing or refractory hypercalcemia with 8 mg zoledronic acid resulted in a 52% complete response rate. Fever, hypophosphatemia, and asymptomatic hypocalcemia were the most common drug-related adverse events. These studies have shown that a short single intravenous dose of 4 mg or 8 mg zoledronic acid is effective in treating moderate to severe HCM. Zoledronic acid produced a higher rate of calcium normalization, faster onset of action, and longer time to relapse than pamidronate, while maintaining an excellent safety profile. The lower dose of 4 mg is recommended as initial therapy, with the 8 mg dose reserved for patients requiring retreatment.

Expression of common acute lymphoblastic leukemia-associated antigen on germ cell tumor.

A mediastinal germ cell tumor is described that reacts with the anti-common acute lymphoblastic leukemia-associated antigen antibody J5 using both immunofluorescence and immunoperoxidase techniques. This antigen has been reported recently on various cell lines including melanoma, colon, and breast. It has also been seen on normal fibroblasts and peripheral granulocytes. This is believed to be the first description of a solid nonlymphoid neoplasm possessing this antigen, and the implications regarding prognosis and therapy are discussed.

Monoclonal antibodies to antigens abnormally expressed in breast cancer.

We report the production, screening, and characterization of ten murine monoclonal antibodies directed at antigens that are expressed abnormally in human breast tumors. Immunoperoxidase staining of frozen and fixed tissues shows the antigens to be present at low levels on the luminal membrane of normal breast cells and at high levels in the cytoplasm and surface membrane of breast tumor cells. The ten antibodies appear to recognize six different epitopes on the basis of their quantitative differences in reactivity against four antigen preparations, as measured by ELISA. Immunoblots show that eight of the ten antibodies recognize a 300,000 MW molecule from breast tumor preparations; six of these antibodies also react with a second molecule from the same tumor preparations of 280,000 MW. Seven antibodies react with an antigen from milk fat globule membrane of 330,000 MW. It therefore appears that the two molecules from tumor tissue and the one molecule from normal tissue share common epitopes. Selected antibodies were tested for reactivity against 25 primary breast tumors and 14 pairs of primary and metastatic breast tumors. Three antibodies have broad reactivity and stain more than 80% of primary tumors; the three other antibodies identify subsets of those tumors. Results of staining pairs of primary and metastatic lesions show that metastases continue to express antigens of the primary lesion in a high percentage of cells.

Instability of (ara-C) DNA under alkaline conditions.

We employed both [5-3H] ara-C and ([2-14C] ara-C labeled L1210 DNA for analysis following exposure to alkali under various conditions. The results demonstrated that the tritium label on C5 of ara-C molecules incorporated in DNA was exchanged with water under alkaline conditions and, therefore, radioactivity was subsequently detectable in the acid-soluble fraction. The [14C] ara-C labeled DNA, however, was not susceptible to loss or radioactivity by this mechanism, and the appearance of this isotope in the acid-soluble fraction required degradation of the DNA strand or pyrimidine ring. Our results indicated that the [14C] labeled DNA was degraded by alkali, suggesting structural instability of this abnormal nucleic acid. These findings provide useful technical information on the purification of (ara-C) DNA labeled with different isotopes.

Effect of ARA-C incorporation on deoxyribonucleic acid synthesis in cells.

Recent work using isolated DNA polymerase-template complexes has shown that arabinofuranosyl derivatives con slow DNA synthesis by being incorporated into DNA. Our results suggest that these agents act by a similar mechanism in L1210 cells. The results demonstrate that inhibition of cellular DNA synthesis by cytosine arabinoside (ara-C) was significantly related to the extent of ara-C incorporation in DNA over a wide range of drug concentrations and times of exposure. Furthermore, treatment with increasing concentrations of ara-C resulted in a greater proportion of ara-C residues at the 3'-terminus of the elongating DNA chain. These observations suggest that ara-C incorporation results in poor primer termini for further chain elongation.

Modulation of 5-FU metabolism in human MCF-7 breast carcinoma cells.

We have previously demonstrated a highly significant relationship (P less than 0.0001) between the incorporation of 5-fluorouracil (5-FU) into total cellular RNA and loss of clonogenic survival of the human MCF-7 breast carcinoma cell line. The present studies explore the applicability of this relationship to MCF-7 cells exposed to 5-FU and modulating agents such as PALA, MTX, and MMPR. PALA treatment produces a minimal increase in the absolute amount of 5-FU incorporated into total cellular RNA, but it results in a three-fold enhancement of the [3H]FU/32P ratio, which measures 5-FU misincorporation into newly synthesized RNA. MTX and MMPR increase intracellular PRPP levels up to four-fold; nevertheless these agents result in only minimal increases in absolute (5-FU)RNA formation. In contrast, the relative incorporation of 5-FU into newly synthesized RNA of MTX- or MMPR-treated cells is increased 2.5-fold. The combination of PALA/MMPR results in a two-fold absolute increase in (5-FU)RNA formation and a nine-fold enhancement of the [3H]FU/32P ratio. Combinations of modulating agents with 5-FU result in more than additive decreases in MCF-7 clonogenic survival. The relationship between 5-FU incorporation into RNA and loss of clonogenic survival was highly significant (P less than 0.0002) when corrected for newly synthesized RNA, while the correlation with absolute amounts of (5-FU)RNA formation was less significant (P less than 0.05). These studies demonstrate that the relationship previously established between (5-FU)RNA formation and loss of clonogenic survival should be corrected for the amount of newly synthesized RNA when 5-FU is combined with modulating agents that alter rates of RNA synthesis.

Deoxycoformycin: neurological toxicity.

Deoxycoformycin (DCF) is a tight-binding inhibitor of adenosine deaminase (ADA) currently undergoing phase I--II evaluation. Neurological toxicity has been a frequent and occasionally severe complication of treatment with this drug. A T-cell leukemia patient with an Ommaya reservoir was treated with DCF, and the pharmacokinetics of the drug in the cerebrospinal fluid and plasma were studied. DCF penetrates the cerebrospinal fluid and achieves levels as high as 1/10 the concurrent plasma levels. The accumulation of adenosine and deoxyadenosine in plasma, cerebrospinal fluid, and urine was monitored; the neuropharmacological effect of these metabolites is discussed.

Recognition of peptidyl epitopes by polymorphic epithelial mucin (PEM)-specific monoclonal antibodies.

Peptidyl epitope recognition by several murine monoclonal antibodies (MAbs E29, H23, HMFG-1, HMFG-2, MA5, MA6 and MA9) which react with the polymorphic epithelial mucins [PEM; epithelial membrane antigen (EMA)] was studied by using ten synthetic peptides representative of the 20 residue tandem repeat as test antigens. Antibody binding to 6-10 residue overlaps and to peptides having a common carboxy-terminus and staggered amino-termini (8-31 residues) was assessed by solid phase and competition ELISA techniques. From these analyses, all MAbs except MA9 were found to react predominantly with the carboxy-terminal half of the repeat motif. Polyclonal antibody responses in mice immunized with intact EMA/PEM-containing preparations also displayed significant reactivities against synthetic repeat peptide antigens and, conversely, synthetic peptides as carrier-conjugated immunogens induced antibodies recognizing intact antigens. These results are discussed vis-à-vis peptide conformation, the potential effects of O-glycosylation on secondary structure, and the possible effects of these parameters on immunogenicity and antigenicity.

Studies on the mechanism of action of cytosine arabinoside.

We have demonstrated that Ara-C incorporates in leukemic cell DNA and that the extent of this incorporation correlates significantly with loss of clonogenic survival. This relationship between formation of (Ara-C)DNA and cytotoxicity is also obtained in the presence of modulating agents such as thymidine. Further, the (Ara-C)DNA is structurally abnormal and undergoes strand scission upon exposure to alkali. We have also demonstrated that the incorporated Ara-C residue serves as a poor primer terminus for further chain elongation and thereby results in inhibition of DNA synthesis. These findings provide new insights into the mechanism of action of the most effective agent in the treatment of acute myelogenous leukemia. These findings have also been extended to demonstrate that Ara-C results in the induction of leukemic cell differentiation as well as lethal cellular events.

5-Fluorouracil incorporation into human breast carcinoma RNA correlates with cytotoxicity.

We demonstrate a highly significant relationship (p less than 0.0001) between the incorporation of 5-flourouracil in total cellular RNA and loss of clonogenic survival of the human MCF-7 breast carcinoma cell line. The extent of 5-fluorouracil incorporation in RNA is concentration- and time-dependent. Identical results are obtained in experiments employing thymidine to bypass the block of thymidylate synthetase and reverse inhibition of DNA synthesis. These studies suggest that the incorporation of 5-fluorouracil in RNA is the major mechanism of cytotoxic action in this human cell line.

Oral bisphosphonates: A review of clinical use in patients with bone metastases.

BACKGROUND: This study was conducted to review the efficacy and safety of oral bisphosphonates for the treatment of bone metastases in cancer patients. METHODS: Available published clinical studies of oral bisphosphonates in bone metastases from 1980 to the present were identified through a MEDLINE search and from literature references. Data were reviewed for efficacy and safety, with an emphasis on double blind, placebo-controlled studies; clinically relevant endpoints; and appropriate study methodology. RESULTS: Etidronate, alendronate, pamidronate, risedronate, and tiludronate currently are available in the U.S. as either intravenous and/or oral formulations. Although newer bisphosphonates are more potent, oral bioavailability remains < 1%. Oral etidronate has been found to be ineffective in patients with multiple myeloma and prostate carcinoma bone metastases. Pamidronate has been found to be effective in reducing skeletal morbidity associated with bone metastases in both multiple myeloma and breast carcinoma patients when given intravenously, but is ineffective orally in multiple myeloma patients. To the authors' knowledge, there are no double blind, placebo-controlled trials of oral pamidronate in patients with breast carcinoma and bone metastases. Several clinical trials with clodronate, a bisphosphonate that is not available in the U.S., have shown mixed results in patients with myeloma and breast carcinoma bone metastases. To the authors' knowledge, there are no published trials evaluating oral alendronate, tiludronate, or risedronate in patients with metastases to bone. CONCLUSIONS: Oral bisphosphonates do not appear to be as effective as intravenous administration in reducing skeletal complications in patients with metastases to bone lesions. Low oral bioavailability is the most likely reason for this difference. Oral dosing should not be substituted for intravenous administration in the treatment of malignant osteolysis.

Enhanced expression and secretion of an epithelial membrane antigen (MA5) in a human mucinous breast tumor line (BT549).

The mouse monoclonal antibody MA5, generated versus a membrane-enriched extract of breast cancer metastatic to liver, detects one or two high molecular weight species (greater than 200 kD) in breast tumor membranes, human milk fat globule membranes, and various breast tumor cell lines. From comparative studies of five breast carcinoma lines (BT20, BT549, MCF-7, T47D, and ZR75-1), as well as an epithelial line established from milk (HBL-100), we report the stimulation of expression of MA5-reactive antigen in a mucinous breast tumor cell line (BT549) through the use of a culture medium supplemented with charcoal-absorbed fetal calf serum, insulin, and hydrocortisone. Large amounts of aggregated MA5-reactive antigen are secreted into the culture medium and can be recovered from the media for further purification by centrifugation. These findings suggest that BT549 cells, grown in the special nutritive medium, may be useful in providing an ample source of epithelial membrane antigen (also termed polymorphic epithelial mucin) for standardization of clinical assay protocols, as well as provide a model system for studies of the regulation of expression for this class of antigens in breast carcinoma.