A randomized phase II study comparing capecitabine alone with capecitabine and oral cyclophosphamide in patients with advanced breast cancer-cyclox II.

BACKGROUND: Capecitabine and cyclophosphamide are active in patients with advanced breast cancer, have non-overlapping toxic effects and synergy pre-clinically. We explored the efficacy and toxic effect of an all-oral combination of capecitabine with cyclophosphamide versus capecitabine alone in a multicentre, randomized, phase II study. PATIENTS AND METHODS: Patients with locally advanced or metastatic breast cancer were randomized to treatment with capecitabine given continuously (666 mg/m(2) b.i.d. days 1-28) alone (C) or with oral cyclophosphamide (100 mg/m(2) days 1-14 of a 28-day cycle) (CCy) for up to six cycles. RESULTS: Eighty-two patients were randomized. There was no complete response. The proportions with partial response were 36% on C and 44% on CCy, a difference of 7.9% [95% confidence interval (CI) -13.4 to 29.1]. Significant toxic effect was uncommon: grade ≥3 diarrhoea in 4 (10%) versus 1 (3%) patients; grade ≥3 fatigue in 2 (5%) versus 5 patients (13%) and grade ≥2 hand-foot syndrome in 7 (17%) versus 11 (28%) patients receiving C versus CCy, respectively. Median progression-free survival was 3.1 months on C and 6.9 months on CCy, not significantly different statistically. There was no difference in overall survival. CONCLUSION: The difference in tumour response suggests a reasonable chance that CCy is superior to C alone.

Different extracellular calcium requirements for proliferation of nonneoplastic, preneoplastic, and neoplastic mouse cells.

The DNA-synthetic and proliferative activities of freshly isolated, nontumorigenic C3H mouse skin cells (first passage) were lowest when the extracellular free (or ionic) calcium level was reduced to between 0.05 and 0.1 mM, whereas the extracellular free calcium level in cultures of repeatedly passaged, preneoplastic C3H/10T1/2 and MCA-C3H/10T1/2 type I mouse fetal fibroblasts had to be reduced to 0.01 mM or less before the DNA-synthetic and proliferative activities were minimal. This inhibition of DNA synthesis and cell multiplication by calcium deprivation was rapidly reversed by returning the extracellular calcium level to its normal value. In contrast, the neoplastic fibrosarcoma-forming, MCA-C3H/10T1/2 type III mouse fetal fibroblasts could synthesize DNA and could multiply indefinitely even in the presence of an extremely low concentration of extracellular free calcium. Thus, the extracellular calcium requirement for DNA synthesis and proliferation appears to reflect the tumorigenic potential of the cell.

Changes in cyclic adenosine monophosphate-responsive element binding proteins in rat hepatomas.

We applied Southwestern and Western blotting and gel retardation techniques to investigate the changes that occur in the cyclic adenosine monophosphate (cAMP)-responsive element (CRE) binding (CREB) proteins in rapidly growing, chemically induced 5123tc and 5123D Morris hepatomas. Using the CRE sequences from the c-fos, E2A, and somatostatin gene promoters, we identified in the nuclear proteins from normal unstimulated or proliferating rat liver cells six different protein factors of Mr 34,000, 36,000, 40,000, 47,000, 56,000, and 72,000 capable of binding to the element. The Mr 47,000 protein had the highest specificity for the core CRE, suggesting its importance in cAMP-mediated gene expression. We could not find the Mr 47,000 CREB protein in the 5123tc and 5123D hepatomas. Our efforts to detect this protein in the tumors by (a) using the CRE sequence from different gene promoters, (b) altering the protocol for extracting nuclear proteins, or (c) attempting to restore its DNA-binding property by phosphorylation [with endogenous protein kinase(s), a catalytic subunit of cAMP-dependent protein kinase, and protein kinase C/dephosphorylation (with alkaline phosphatase)] were unsuccessful. The loss of tje Mr 47,000 CREB protein from solid tumors of the Morris hepatoma is likely to be related to the neoplastic properties of the tumor cell rather than to cell growth because the level of this protein remained unchanged during a 6-day period of liver regeneration. The nuclear extract from the Morris hepatoma that did not have the Mr 47,000 CRE-binding factor contained proteins immunologically related to the CREB, c-Jun, and c-Fos proteins. We conclude that the Mr 47,000 factor represents a distinct member of the CRE-binding protein family and that its absence from the hepatomas may lead to aberrant expression of cAMP-inducible genes.

Physiological regulation of eukaryotic topoisomerase II.

Topoisomerase II is an essential enzyme in all organisms with several independent roles in DNA metabolism. In this article we review our knowledge on the regulation of the expression and catalytic activity of topoisomerase II in both lower and higher eukaryotes. Current data indicate that the regulation of topoisomerase II gene expression is complex, with positive and negative controls in evidence at the level of both promoter activity and mRNA stability. Similarly, the activity of the mature enzyme can be regulated by the action of several different protein kinases. Of particular interest is the cell cycle-dependent phosphorylation of topoisomerase II, including multiple, mitosis-specific modifications, which are proposed to regulate the essential chromosome decatenation activity of the enzyme.

Relationship of DNA structure to internal dynamics: correlation of helical parameters from NOE-based NMR solution structures of d(GCGTACGC)(2) and d(CGCTAGCG)(2) with (13)C order parameters implies conformational coupling in dinucleotide units.

The coupling between the conformational properties of double-stranded DNA and its internal dynamics has been examined. The solution structures of the isomeric DNA oligomers d(GCGTACGC)(2) (UM) and d(CGCTAGCG)(2) (CTSYM) were determined with (1)H NMR spectroscopy by utilizing distance restraints from total relaxation matrix analysis of NOESY cross-peak intensities in restrained molecular dynamics calculations. The root-mean-square deviation of the coordinates for the ensemble of structures was 0.13 A for UM and 0.49 A for CTSYM, with crystallographic equivalent R(c)=0.41 and 0.39 and sixth-root residual R(x)=0.11 and 0.10 for UM and CTSYM, respectively. Both UM and CTSYM are B-form with straight helical axes and show sequence-dependent variations in conformation. The internal dynamics of UM and CTSYM were previously determined by analysis of (13)C relaxation parameters in the context of the Lipari & Szabo model-free formalism. Helical parameters for the two DNA oligomers were examined for linear correlations with the order parameters (S(2)) of groups of (13)C spins in base-pairs and dinucleotide units of UM and CTSYM. Correlations were found for six interstrand base-pair parameters tip, y-displacement, inclination, buckle and stretch with various combinations of S(2) for atoms in Watson-Crick base-pairs and for two inter-base-pair parameters, rise and roll with various combinations of S(2) for atoms in dinucleotides. The correlations for the interstrand base-pair helical parameters indicate that the conformations of the deoxyribose residues of each strand are dynamically coupled. Also, the inter-base-pair separation has a profound effect on the local internal motions available to the DNA, supporting the idea that rise is a principal degree of freedom for DNA conformational variability. The correlations indicate collective atomic motions of spins that may represent specific motional modes in DNA, and that base sequence has a predictable effect on the relative order of groups of spins both in the bases and in the deoxyribose ring of the DNA backbone. These observations suggest that an important functional outcome of DNA base sequence is the modulation of both the conformation and dynamic behavior of the DNA backbone.

NMR evidence for mechanical coupling of phosphate B(I)-B(II) transitions with deoxyribose conformational exchange in DNA.

The conformational exchange of the phosphate and deoxyribose groups of the DNA oligomers d(GCGTACGC)(2) and d(CGCTAGCG)(2) have been investigated using a combination of homonuclear and heteronuclear NMR techniques. Two-state exchange between phosphate B(I) and B(II) conformations and deoxyribose N and S conformations was expressed as percent population of the major conformer, %B(I) or %S. Sequence context-dependent variations in %B(I) and %S were observed. The positions of the phosphate and deoxyribose equilibria provide a quantitative measure of the ps to ns timescale dynamic exchange processes in the DNA backbone. Linear correlations between %B(I), %S, and previously calculated model free (13)C order parameters (S(2)) were observed. The %B(I) of the phosphates were found to be correlated to the S(2) of the flanking C3' and C4' atoms. The %B(I) was also found to be correlated with the %S and C1' S(2) of the deoxyribose ring 5' of the phosphates. The %B(I) of opposing phosphates is correlated, while the %B(I) of sequential phosphates is anti-correlated. These correlations suggest that conformational exchange processes in DNA are coupled to each other and are modulated by DNA base sequence, which may have important implications for DNA-protein interactions.

Lactam formation increases receptor binding, adenylyl cyclase stimulation and bone growth stimulation by human parathyroid hormone (hPTH)(1-28)NH2.

Human parathyroid hormone (1-28)NH2 [hPTH(1-28)NH2] is the smallest of the PTH fragments that can fully stimulate adenylyl cyclase in ROS 17/2 rat osteoblast-like osteosarcoma cells. This fragment has an IC50 of 110 nM for displacing 125I-[Nle8,18,Tyr34]bovine PTH(1-34)NH2 from HKRK B7 porcine kidney cells, which stably express 950,000 human type 1 PTH/PTH-related protein (PTHrP) receptors (PTH1Rs) per cell. It also has an EC50 of 23.9 nM for stimulating adenylyl cyclase in ROS 17/2 cells. Increasing the amphiphilicity of the alpha-helix in the residue 17-28 region by replacing Lys27 with Leu and stabilizing the helix by forming a lactam between Glu22 and Lys26 to produce the [Leu27]cyclo(Glu22-Lys26)hPTH(1-28)NH2 analog dramatically reduced the IC50 for displacing 125I-[Nle8,18,Tyr34]bPTH(1-34)NH2 from hPTH1Rs from 110 to 6 nM and dropped the EC50 for adenylyl cyclase stimulation in ROS 17/2 cells from 23.9 to 9.6 nM. These modifications also increased the osteogenic potency of hPTH(1-28)NH2. Thus, hPTH(1-28)NH2 did not significantly stimulate either femoral or vertebral trabecular bone growth in rats when injected daily at a dose of 5 nmol/100 g body weight for 6 weeks, beginning 2 weeks after ovariectomy (OVX), but it strongly stimulated the growth of trabeculae in the cancellous bone of the distal femurs and L5 vertebrae when injected at 25 nmol/100 g body weight. By contrast [Leu27]cyclo(Glu22-Lys26)hPTH(1-28)NH2 significantly stimulated trabecular bone growth when injected at 5 nmol/100 g of body weight. Thus, these modifications have brought the bone anabolic potency of hPTH(1-28)NH2 considerably closer to the potencies of the larger PTH peptides and analogs.

Parathyroid hormone fragments may stimulate bone growth in ovariectomized rats by activating adenylyl cyclase.

PTH is regarded conventionally as a catabolic hormone that stimulates osteoclastic resorption of bone. However, it has been known since 1932 that intermittent pulses of PTH stimulate bone formation in animals and humans. PTH independently activates two signal mechanisms: one that stimulates adenylyl cyclase and one that stimulates protein kinase C (PKC). The goal of this study was to use the 3- to 5-month-old ovariectomized (OVX) rat model to determine which of the two signal mechanisms is responsible for the anabolic action of PTH on bone. OVX triggered a large loss of trabecular bone without significantly affecting the normal slow growth of cortical bone in the distal halves of the femora. Daily injections of human hPTH(1-34) fragment (1 nmol/100 g body weight), which stimulated both adenylyl cyclase and membrane-associated PKC activity in osteoblast-like ROS 17/2 rat osteosarcoma cells, stimulated the growth of both cortical and trabecular bone in the OVX rats. Daily injections of the same dose of hPTH(1-31), which stimulated adenylyl cyclase but not PKC in ROS 17/2 cells, stimulated trabecular bone growth in the OVX rats less effectively than hPTH(1-34), but it stimulated cortical bone growth as rapidly and as dramatically as hPTH(1-34). Injections of equimolar amounts of desamino-hPTH(1-34) [N-propionyl(2-3)hPTH-amide], which stimulated PKC as strongly as hPTH(1-34) in ROS 17/2 cells but had a drastically reduced ability to stimulate adenylyl cyclase, or injections of recombinant hPTH(8-84) which stimulated PKC only in the ROS 17/2 cells, did not stimulate cortical or trabecular bone growth in the OVX animals. Thus, cyclic AMP and cyclic AMP-dependent protein kinases may be the primary mediators of the anabolic action of intermittent pulses of PTH on bone in OVX rats.

Stimulation of protein kinase C activity in cells expressing human parathyroid hormone receptors by C- and N-terminally truncated fragments of parathyroid hormone 1-34.

The parathyroid hormone (PTH) fragment PTH(1-34) stimulates adenylyl cyclase, phospholipase C (PLC), and protein kinase C's (PKCs) in cells that express human, opossum, or rodent type 1 PTH/PTH-related protein (PTHrP) receptors (PTHR1s). Certain carboxyl (C)-terminally truncated fragments of PTH(1-34), such as human PTH(1-31) [hPTH-(1-31)NH2], stimulate adenylyl cyclase but not PKCs in rat osteoblasts or PLC and PKCs in mouse kidney cells. The hPTH(1-31)NH2 peptide does fully stimulate PLC in HKRK B7 porcine renal epithelial cells that express 950,000 transfected hPTHR1s per cell. Amino (N)-terminally truncated fragments, such as bovine PTH(3-34) [bPTH(3-34)], hPTH(3-34)NH2, and hPTH(13-34), stimulate PKCs in Chinese hamster ovary (CHO) cells expressing transfected rat receptors, opossum kidney cells, and rat osteoblasts, but an intact N terminus is needed to stimulate PLC via human PTHR1s in HKRK B7 cells. We now report that the N-terminally truncated analogs bPTH(3-34)NH2 and hPTH(13-34)OH do activate PKC via human PTHR1s in HKRK B7 cells, although less effectively than hPTH(1-34)NH2 and hPTH(1-31)NH2. Moreover, in a homologous human cell system (normal foreskin fibroblasts), these N-terminally truncated fragments stimulate PKC activity as strongly as hPTH(1-34)NH2 and hPTH(1-31)NH2. Thus, it appears that unlike their opossum and rodent equivalents, hPTHR1s can stimulate both PLC and PKCs when activated by C-terminally truncated fragments of PTH(1-34). Furthermore, hPTHR1s, like the PTHR1s in rat osteoblasts, opossum kidney cells, and rat PTHR1-transfected CHO cells also can stimulate PKC activity by a mechanism that is independent of PLC. The efficiency with which the N-terminally truncated PTH peptides stimulate PKC activity depends on the cellular context in which the PTHR1s are expressed.

The protein kinase-C activation domain of the parathyroid hormone.

The PTH activates both adenylate cyclase and a mechanism that increases membrane-associated protein kinase-C (PKC) activity. To define the hormone's PKC activation domain we have used a panel of PTH fragments and ROS 17/2 rat osteosarcoma cells as the target cells. PTH equally and maximally increased PKC activity in ROS 17/2 cell membranes at physiological concentrations between 1-50 pM and 5-50 nM, but not at intermediate concentrations or concentrations above 50 nM. The PKC-stimulating picomolar concentrations of PTH did not stimulate adenylate cyclase in ROS 17/2 cells, while the PKC-stimulating nanomolar concentrations of the hormone did stimulate adenylate cyclase, with an EC50 of 1-2 nM. Very high concentrations of PTH, such as 100 nM, that did not increase membrane PKC activity were still able to maximally stimulate adenylate cyclase. PTH fragments lacking the N-terminal amino acids needed for adenylate cyclase activation increased membrane PKC activity, and the PKC activation domain was found to lie within the 28-34 region of the PTH molecule. This was confirmed by showing that optimally effective picomolar concentrations of the human PTH-(28-34) fragment itself were able to increase membrane-associated PKC activity to the same extent as the optimally effective picomolar concentrations of the intact PTH-(1-84) or the larger PTH-(1-34) or PTH-(3-34) fragments.

Prolonged low-dose infusion of human parathyroid hormone does not increase femoral cancellous bone volume in ovariectomized rats.

OBJECTIVE: Daily injections of human parathyroid hormone (hPTH) increase bone volume in various animal species and in osteoporotic women. For hPTH to be widely accepted as an anabolic therapy for treating postmenopausal osteoporosis alternative delivery options need to be explored to replace the need for daily patient subcutaneous self-injection. Among these are inhalation, oral delivery and the use of programmable implanted minipumps to deliver the peptide. While infusion of high doses of PTH causes bone loss and hypercalcemia, no studies have assessed the effects of prolonged infusion of low doses of PTH on bone growth. DESIGN AND METHODS: [Leu(27)]-cyclo(Glu(22)-Lys(26))-hPTH-(1--31)NH(2) was delivered by Alzet minipumps to ovariectomized rats for 6 weeks after which histomorphometric indices (cancellous bone volume, trabecular thickness, mean trabecular number) of bone formation were measured in distal femurs. RESULTS: Infusing low doses (0.05 and 0.1 nmole/100g body weight/day) of the hPTH analog, [Leu(27)]-cyclo(Glu(22)-Lys(26))-hPTH-(1--31)NH(2), for 6 weeks does not prevent the ovariectomy-induced loss of rat femoral cancellous bone volume, trabecular thickness or trabecular number. CONCLUSION: These results support the absolute requirement of daily injections for the osteogenic action of hPTH on bone.

Regulation of the human topoisomerase IIalpha gene promoter in confluence-arrested cells.

Expression of DNA topoisomerase IIalpha mRNA and protein reflects the proliferative state of mammalian cell lines and tissues with high levels in actively cycling cells but marked down-regulation during serum deprivation or cell density-induced growth arrest. Using stably integrated gene fusions comprising the human topoisomerase IIalpha promoter with a growth hormone reporter gene, we have localized elements required for the differential activity of the topoisomerase IIalpha promoter in proliferating and confluence-arrested cells. Deletion analysis localized the region of the promoter that responded to changes in the cellular growth state to between -101 and -144 base pairs. Mutation analysis identified an inverted CCAAT box (ICB) located at -108 to -104 as necessary for promoter down-regulation in confluence-arrested cells, while several other potential cis-acting elements, including four additional ICBs, were shown not to be required. The critical ICB was recognized in vitro by the CCAAT box binding factor, NF-Y, with levels of binding activity higher in extracts from proliferating cells than from confluence-arrested cells. We conclude that the differential regulation of topoisomerase IIalpha gene expression in cycling and confluence-arrested cells is mediated, at least in part, through proliferation-specific binding of factors to an ICB element in the gene promoter.

A possible involvement polyamines in the initiation of DNA synthesis by human WI-38 and mouse BALB/3T3 cells.

Changing the medium, or adding fresh serum, induces a large proportion of the proliferatively quiescent cells in confluent monolayers of human WI-38 and mouse BALB/3T3 cells to initiate a growth-division cycle. Exposure at the time of the medium change or serum addition to MGBG (methyl glyoxal bis [guanylhydrazone]), an inhibitor of spermidine and spermine synthesis and function, reduces or stops the subsequent flow of cells into the DNA-synthetic phase, without grossly affecting RNA synthesis. Mediation of MGBG action by an actual or functional shortage of spermidine or spermine (but not putrescine), and consequently an involvement of these polyamines in DNA synthesis, is strongly suggested by the reduction of the inhibitor's effectiveness by a brief (1-hour), early prereplicative exposure of the treated cells to exogenous spermidine and spermine (but not putrescine).

Control of DNA polymerase-alpha activity in regenerating rat liver by calcium and 1 alpha,25(OH)2D3.

The late G1 surge of DNA polymerase-alpha activity and the initiation of DNA replication in the hepatocytes of partial hepatectomy-induced regenerating liver were severely reduced when the mitogenic partial hepatectomy was carried out in the hypocalcemic and 1,25(OH)2D3 (1 alpha,25-dihydroxycholecalciferol)-deficient environment of parathyroidectomized (PTX) or thyroparathyroidectomized (TPTX) rats. These inhibitions were prevented in TPTX rats by a postpartial hepatectomy injection of 1,25(OH)2D3, which also restored blood calcium to normocalcemic levels. Inhibition of active DNA polymerase-alpha accumulation and initiation of DNA synthesis in TPTX rats were also completely prevented by prefeeding the rats a low phosphorus diet, which stopped the lowering of the blood levels of calcium and 1,25(OH)2D3 following parathyroid removal. These studies indicate that the rise of DNA polymerase-alpha activity and the initiation of DNA replication in regenerating liver are controlled by cellular processes that rely on normal blood levels of calcium and 1,25(OH)2D3. Because DNA polymerase-alpha is the third DNA replication enzyme (the others are ribonucleotide reductase and thymidylate synthase) that has been shown to depend on parathyroid hormone and/or the circulating levels of calcium and 1,25(OH)2D3 that it controls, the authors concluded that the processes dependent on calcium and 1,25(OH)2D3 are parts of a mechanism that coordinately activates the DNA-replicating enzymes. The possibility that cyclic adenosine monophosphate (cAMP)-dependent protein kinases are involved in this replication mechanism is considered.

Tamoxifen as systemic treatment of advanced breast cancer during pregnancy--case report and literature review.

BACKGROUND: When patients with metastatic breast cancer become pregnant, management is complicated by the potential harms of drug treatment to the fetus and by the potential effects of the pregnancy on the cancer. Chemotherapy is considered optimal systemic anti-cancer therapy from the second trimester, while tamoxifen has been considered inappropriate due to concerns over possible teratogenesis and lack of efficacy. CASE: We report a patient who became pregnant concurrent with the identification of metastatic breast cancer and who elected to continue her pregnancy with tamoxifen as sole systemic anti-cancer therapy. The pregnancy was difficult, but a normal child was delivered and the mother responded to subsequent hormone manipulation. The putative teratogenic effects of tamoxifen and the mechanisms underlying tamoxifen resistance in this setting are discussed. CONCLUSIONS: The use of tamoxifen in pregnancy is complex, but is not necessarily associated with fetal harm and may be considered a therapeutic option in selected cases.

Calcium-dependent stimulation of BALB/c 3T3 mouse cell DNA synthesis by a tumor-promoting phorbol ester (PMA).

During the first six passages after their arrival in this laboratory, BALB/c 3T3 mouse cells did not proliferate in serum containing-medium having an ionic calcium concentration of 0.05 mM or less, but by the ninth passage they had become able to multiply in the presence of these lower calcium levels. In low calcium (e.g., 0.02 mM) medium, passage 1-6, cells in sparse cultures were blocked at the Gl/S boundary of their cycle. These blocked cells could be induced to start making DNA within only one hour either by returning the ionic calcium level to a normal range of values (1.25 mM), or by adding 0.05 mug/ml of PMA (12-O-tetradecanoyl-phorbol-13-acetate). PMA probably acted by sensitizing the blocked cells to calcium rather than replacing the ion, because it was ineffective in ionic calcium-free medium. Finally, PMA did not by itself induce proliferation of cells (regardless of the number of passages) which had been proliferatively inactivated by density-dependent factors in confluent cultures. However, PMA did promote DNA synthesis by these cells during their brief transition to the "cycling" state caused by exposure to fresh serum-containing medium.