Urinary and fecal incontinence in patients with advanced ovarian cancer treated with CRS + HIPEC.

PURPOSE: The objective of this work was to analyze the long-term prevalence of urinary and fecal incontinence and their impact on quality of life in patients with advanced and recurrent ovarian cancer treated with cytoreductive surgery and hyperthermic intraoperative intraperitoneal chemotherapy (CRS + HIPEC). METHODS: This cross-sectional study included a series of patients with advanced and recurrent ovarian cancer treated by CRS + HIPEC, with a disease-free period of at least 12 months after the procedure. Urinary incontinence was evaluated using the International Consultation on Incontinence Questionnaire - Short Form (ICIQ-SF), fecal incontinence using the Wexner test and the Fecal Incontinence Quality of Life (FIQL) questionnaire and global quality of life using the Short Form 36 (SF-36) survey. RESULTS: A total of 64 patients were included in the study, with a median age of 55 years (range 28-78). The urinary incontinence rate was 45% and the fecal incontinence rate was 20%. Up to 14% of the patients presented both types of incontinence. The presence of urinary or fecal incontinence generated a significant negative impact on quality of life in relation to patients without incontinence. DISCUSSION: Urinary and fecal incontinence is frequent in the follow-up of ovarian cancer patients treated with CRS + HIPEC. Reconsidering the approach to the pelvis without peritoneal metastases in the peritoneum could modify the incidence of these pelvic floor dysfunctions.

Risk factors and management of incisional hernia after cytoreduction and hyperthermic intraperitoneal chemotherapy (HIPEC) in patients with peritoneal surface malignancies.

BACKGROUND: The incidence of incisional hernia in patients with peritoneal surface malignancies treated by cytoreduction plus hyperthermic intraperitoneal chemotherapy (HIPEC) remains unclear, and the criteria commonly used to indicate their repair cannot be applied in these patients. The objective of this work was to analyze the incidence of incisional hernias in these patients, identify the risk factors associated with their appearance, and propose an algorithm for their management. METHODS: We analyzed a series of patients with malignant pathologies of the peritoneal surface treated by cytoreduction with peritonectomy and HIPEC procedures between January 2008 and June 2017. Only patients with a minimum postoperative follow-up period of 12 months were included. RESULTS: Our series included 282 patients, 28 (10%) of whom developed an incisional hernia during the follow-up period. Fifty-one patients, all with ovarian cancer with peritoneal dissemination, did not receive HIPEC after cytoreduction as they were part of the control arm of the CARCINOHIPEC clinical trial (NCT02328716) or because they did not provide specific informed consent. In the multivariate analysis, treatment with HIPEC (OR 2.56, 95% CI [1.57, 4.31], p = 0.032) and the administration of preoperative systemic chemotherapy (OR = 1.59, 95% CI [1.26, 3.58], p = 0.041) were found to be independent factors related to the appearance of an incisional hernia. CONCLUSIONS: The incidence of incisional hernia after cytoreduction and HIPEC is within the ranges described in the literature for other abdominal surgery procedures. The use of systemic chemotherapy and treatment with HIPEC, in particular, were identified as factors related to their occurrence.

Epigenetic regulation of ID4 in the determination of the BRCAness phenotype in breast cancer.

BRCAness breast tumors represent a group of sporadic tumors characterized by a reduction in BRCA1 gene expression. As BRCA1 is involved in double-strand breaks (DSBs) repair, dysfunctional BRCA pathway could make a tumor sensitive to DNA damaging drugs (e.g., platinum agents). Thus, accurately identifying BRCAness could contribute to therapeutic decision making in patients harboring these tumors. The purpose of this study was to identify if BRCAness tumors present a characteristic methylation profile and/or were related to specific clinico-pathological features. BRCAness was measured by MLPA in 63 breast tumors; methylation status of 98 CpG sites within 84 cancer-related genes was analyzed by MS-MLPA. Protein and mRNA expressions of the selected genes were measured by quantitative real-time PCR and Western Blot. BRCAness was associated with younger age, higher nuclear pleomorphism, and triple-negative (TN) status. Epigenetically, we found that the strongest predictors for BRCAness tumors were the methylations of MLH1 and PAX5 plus the unmethylations of CCND2 and ID4. We determined that ID4 unmethylation correlated with the expression levels of both its mRNA and protein. We observed an inverse relation between the expressions of ID4 and BRCA1. To the best of our knowledge, this is the first report suggesting an epigenetic regulation of ID4 in BRCAness tumors. Our findings give new information of BRCAness etiology and encourage future studies on potential drug targets for BRCAness breast tumors.

Synthesis and evaluation of hybrid molecules targeting the vinca domain of tubulin.

Some hybrids of vinca alkaloids and phomopsin A, linked by a glycine pattern, have been synthesized in one or two steps, by an insertion reaction and shown to inhibit microtubule assembly. These compounds have been elaborated in order to interact with both the "vinca site" and the "peptide site" of the vinca domain in tubulin. Two out of three hybrids are potent inhibitors of microtubules assembly and they present good cytotoxicity against different cell lines. Molecular modelling studies show that they could bind, within the vinca domain, in similar spatial regions as those of vinca and phomopsin thanks to the flexibility provided by the glycine linker used to elaborate these hybrids.

The pyrimidine nucleoside phosphorylase of Mycoplasma hyorhinis and how it may affect nucleoside-based therapy.

Mycoplasmas are opportunistic parasites and some species are suggested to preferentially colonize tumor tissue in cancer patients. We could demonstrate that the annotated thymidine phosphorylase (TP) gene in the genome of Mycoplasma hyorhinis encodes a pyrimidine nucleoside phosphorylase (PyNPHyor) that not only efficiently catalyzes thymidine but also uridine phosphorolysis. The kinetic characteristics of PyNPHyor-catalyzed nucleoside and nucleoside analogue (NA) phosphorolysis were determined. We demonstrated that the expression of such an enzyme in mycoplasma-infected cell cultures dramatically alters the activity of various anticancer/antiviral NAs such as 5-halogenated pyrimidine nucleosides, including 5-trifluorothymidine (TFT). Due to their close association with human cancers, the presence of mycoplasmas may markedly influence the therapeutic efficiency of nucleoside-based drugs.

Methylation profile of triple-negative breast carcinomas.

Breast cancer is a group of clinically, histopathologically and molecularly heterogeneous diseases, with different outcomes and responses to treatment. Triple-negative (TN) breast cancers are defined as tumors that lack the expression of estrogen receptor, progesterone receptor and epidermal growth factor receptor 2. This subgroup accounts for 15% of all types of breast cancer and its prevalence is higher among young African, African-American and Latino women. The hypermethylation of CpG islands (CpGI) is a common epigenetic alteration for suppressing gene expression in breast cancer and has been shown to be a key factor in breast carcinogenesis. In this study we analyzed the hypermethylation of 110 CpGI within 69 cancer-related genes in TN tumors. For the methylation analysis, we used the methyl-specific multiplex-ligation probe amplification assay. We found that the number of methylated CpGI is similar between TN and non-TN tumors, but the methylated genes between the groups are different. The methylation profile of TN tumors is defined by the methylation of five genes (that is, CDKN2B, CD44, MGMT, RB and p73) plus the non-methylation of 11 genes (that is, GSTP1, PMS2, MSH2, MLH1, MSH3, MSH6, DLC1, CACNA1A, CACNA1G, TWIST1 and ID4). We conclude that TN tumors have a specific methylation profile. Our findings give new information for better understanding tumor etiology and encourage future studies on potential drug targets for triple-negative breast tumors, which now lack a specific treatment.

PM01183, a new DNA minor groove covalent binder with potent in vitro and in vivo anti-tumour activity.

BACKGROUND AND PURPOSE: PM01183 is a new synthetic tetrahydroisoquinoline alkaloid that is currently in phase I clinical development for the treatment of solid tumours. In this study we have characterized the interactions of PM01183 with selected DNA molecules of defined sequence and its in vitro and in vivo cytotoxicity. EXPERIMENTAL APPROACH: DNA binding characteristics of PM01183 were studied using electrophoretic mobility shift assays, fluorescence-based melting kinetic experiments and computational modelling methods. Its mechanism of action was investigated using flow cytometry, Western blot analysis and fluorescent microscopy. In vitro anti-tumour activity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the in vivo activity utilized several human cancer models. KEY RESULTS: Electrophoretic mobility shift assays demonstrated that PM01183 bound to DNA. Fluorescence-based thermal denaturation experiments showed that the most favourable DNA triplets providing a central guanine for covalent adduct formation are AGC, CGG, AGG and TGG. These binding preferences could be rationalized using molecular modelling. PM01183-DNA adducts in living cells give rise to double-strand breaks, triggering S-phase accumulation and apoptosis. The potent cytotoxic activity of PM01183 was ascertained in a 23-cell line panel with a mean GI(50) value of 2.7 nM. In four murine xenograft models of human cancer, PM01183 inhibited tumour growth significantly with no weight loss of treated animals. CONCLUSIONS AND IMPLICATIONS: PM01183 is shown to bind to selected DNA sequences and promoted apoptosis by inducing double-strand breaks at nanomolar concentrations. The potent anti-tumour activity of PM01183 in several murine models of human cancer supports its development as a novel anti-neoplastic agent.

Identification of aspartic acid-203 in human thymidine phosphorylase as an important residue for both catalysis and non-competitive inhibition by the small molecule "crystallization chaperone" 5'-O-tritylinosine (KIN59).

Thymidine phosphorylase (TP) is a catabolic enzyme in thymidine metabolism that is frequently upregulated in many solid tumors. Elevated TP levels are associated with tumor angiogenesis, metastasis and poor prognosis. Therefore, the use of TP inhibitors might offer a promising strategy for cancer treatment. The tritylated inosine derivative 5'-O-tritylinosine (previously designated KIN59) is a non-competitive inhibitor of TP which was previously found to be instrumental for the crystallization of human TP. A combination of computational studies including normal mode analysis, automated ligand docking and molecular dynamics simulations were performed to define a plausible binding site for 5'-O-tritylinosine on human TP. A cavity in which 5'-O-tritylinosine could fit was identified in the vicinity of the Gly405-Val419 loop at a distance of about 11A from the substrate-binding site. In the X-ray crystal structure, this pocket is characterized by an intricate hydrogen-bonding network in which Asp203 was found to play an important role to afford the loop stabilization that is required for efficient enzyme catalysis. Site-directed mutagenesis of this amino acid residue afforded a mutant enzyme with a severely compromised catalytic efficiency (V(max)/K(m) of mutant enzyme approximately 50-fold lower than for wild-type TP) and pronounced resistance to the inhibitory effect of 5'-O-tritylinosine. In contrast, the D203A mutant enzyme kept full sensitivity to the competitive inhibitors 6-aminothymine and 6-amino-5-bromouracil, which is in line with the kinetic properties of these inhibitors. Our findings reveal the existence of a previously unrecognized site in TP that can be targeted by small molecules to inhibit the catalytic activity of TP.

Primary tumour expression of the cysteine cathepsin inhibitor Stefin A inhibits distant metastasis in breast cancer.

Using the clinically relevant 4T1-derived syngeneic murine model of spontaneous mammary metastasis to bone, we have identified the cysteine cathepsin inhibitor Stefin A as a gene differentially expressed in primary and metastatic mammary tumours. In primary tumours, Stefin A expression correlated inversely with metastatic potential in 4T1-derived lines and was not detected in tumour cells in culture, indicating induction only within the tumour microenvironment. Enforced expression of Stefin A in the highly metastatic 4T1.2 cell line significantly reduced spontaneous bone metastasis following orthotopic injection into the mammary gland. Consistent with the mouse data, Stefin A expression correlated with disease-free survival (absence of distant metastasis) in a cohort of 142 primary tumours from breast cancer patients. This was most significant for patients with invasive ductal carcinoma expressing Stefin A, who were less likely to develop distant metastases (log rank test, p = 0.0075). In a multivariate disease-free survival analysis (Cox proportional hazards model), Stefin A expression remained a significant independent prognostic factor in patients with invasive ductal carcinoma (p = 0.0014), along with grade and progesterone receptor (PR) status. In human lung and bone metastases, we detected irregular Stefin A staining patterns, with expression often localizing to micrometastases (<0.2 mm) in direct contact with the stroma. We propose that Stefin A, as a cysteine cathepsin inhibitor, may be a marker of increased cathepsin activity in metastases. Using immunohistology, the cathepsin inhibitor was detected co-expressed with cathepsin B in lung and bone metastases in both the murine model and human tissues. We conclude that Stefin A expression reduces distant metastasis in breast cancer and propose that this may be due to the inhibition of cysteine cathepsins, such as cathepsin B.

Predictive value of apoptosis, proliferation, HER-2, and topoisomerase IIalpha for anthracycline chemotherapy in locally advanced breast cancer.

PURPOSE: Laboratory evidence indicates that tumor growth depends on the balance between cell proliferation and cell death, and many anticancer agents may exert their therapeutic effect by decreasing proliferation and increasing apoptosis. Additionally, clinical observations indicate that overexpression of HER-2 or topoisomerase IIalpha (topo IIalpha) may be predictors of better response to anthracyclines in breast cancer. The objective of this study was to determine if proliferation (Ki-67), apoptosis (TUNEL), and expression of HER-2 and topo IIalpha are affected by anthracycline treatment, and if these molecular markers predict anthracycline responsiveness. EXPERIMENTAL DESIGN: Thirty-three women with primary breast tumors > or =3 cm received either doxorubicin (75 mg/m(2)) or epirubicin (120 mg/m(2)) for 4 cycles before surgery. Clinical response was evaluated after 4 cycles of treatment. Changes in molecular markers were assessed from core needle taken before treatment (D0), at 24-48 h (Dl) and on day 7 (D7) while on treatment, and from the surgical specimen excised on day 84 (D84) after the fourth cycle of chemotherapy. RESULTS: The overall response rate was 51% (17 of 33 patients), with a 12% complete clinical response rate (4 of 33 patients). There were trends for tumors with higher apoptosis and topo IIalpha at baseline (D0) to be more responsive to anthracyclines, p = 0.1 and p = 0.08, respectively. Median apoptosis increased from D0 to Dl (p = 0.06) while median Ki-67 decreased (p = 0.07). Overall, expression of HER-2 remained stable throughout the chemotherapy administration. By Day 84, topo IIalpha had significantly decreased from baseline in responders, while it increased in non-responders, p = 0.03. CONCLUSIONS: In human primary breast cancer, anthracycline treatment causes an early increase in apoptosis and a decrease in proliferation. In this pilot study, higher apoptosis and topo IIalphaa levels in primary tumors were associated with greater responsiveness to anthracyclines, and topo IIalpha levels declined in responsive tumors.

Advances in the chemistry and pharmacology of ecteinascidins, a promising new class of anti-cancer agents.

Ecteinascidins are marine natural products consisting of two or three linked tetrahydroisoquinoline subunits and an active carbinolamine functional group. Their potent antiproliferative activity against a variety of tumor cells has made them attractive candidates for development as anticancer agents. The lead compound, ecteinascidin 743 (ET 743), is currently in phase II clinical trials but the low amounts present in its natural source, the tunicate Ecteinascidia turbinata, made it necessary to develop efficient synthetic procedures. Recent improvements on the original synthesis are reviewed as well as new strategies starting from readily available cyanosafracin B. ET 743 is known to bind to the minor groove of DNA giving rise to a covalent adduct with the exocyclic amino group at position 2 of a guanine in a fashion similar to saframycin antibiotics. Some of the resulting complexes have been studied by a variety of biochemical and spectroscopic methods and also by computer simulations. The rules for sequence specificity have been well established (preferred targets are RGC and YGG, where R and Y stand for purine and pyrimidine, respectively), and it has been shown that binding of ET 743 to DNA is accompanied by minor groove widening and DNA bending towards the major groove. Although the precise target for antitumor action remains to be unambiguously defined, a role in affecting the transcriptional regulation of some inducible genes is rapidly emerging.

Recognition elements that determine affinity and sequence-specific binding to DNA of 2QN, a biosynthetic bis-quinoline analogue of echinomycin.

Footprinting experiments with DNase I provide a starting-point for investigating the molecular basis of nucleotide sequence recognition by 2QN, a bis-quinoline derivative of the quinoxaline antibiotic echinomycin produced by directed biosynthesis in Streptomyces echinatus. Using tyrT DNA molecules variously substituted with inosine and/or 2,6-diaminopurine residues it is shown that the location of the 2-amino group of purine nucleotides in the minor groove of the double helix exerts a dominant influence in determining where the antibiotic will bind, as it does for echinomycin. However, newly created binding sites in DNA molecules substituted with diaminopurine (D), all located round TpD steps, bind 2QN with so much higher affinity than the canonical CpG steps that the latter fail completely to appear as footprints in D-substituted DNA; indeed CpG sequences appear in regions of enhanced susceptibility to nuclease cleavage as do CpI steps in doubly D + I-substituted DNA. Quantitative footprinting plots confirm that sequences surrounding TpD steps bind 2QN several hundred-fold more tightly than do CpG-containing sequences, with dissociation constants of the order of 25 nM. To test the hypothesis that differences in stacking interactions between the chromophores of the drug and the DNA base pairs could account for the differences in binding affinities, models of 2QN bound to two DNA hexamers containing either a central CpG or a central TpD step were built. Calculation of the molecular electrostatic potential (MEP) of 2QN in solution using a continuum method revealed a distinctive pattern that is considered relevant to DNA binding. When the MEPs calculated for the two DNA hexamers in the complexed state were compared, substantial differences were found in the major groove and in the space between the base pairs that is occupied by the chromophores of the drug upon binding. The modelling data support the notion that electrostatic stacking interactions underlie the considerably preferred binding of echinomycin and 2QN around TpD steps rather than CpG steps.

c-erbB-2 (HER-2/neu) protein and drug resistance in breast cancer patients treated with induction chemotherapy.

Expression of c-erbB-2 protein has been associated with poor prognosis and poor response to chemotherapy in breast cancer patients. In the present prospective study, we have analyzed whether c-erbB-2, p53 and P170 proteins may be determinants of tumor resistance in locally advanced breast cancer patients treated with induction chemotherapy. Biopsies (n = 60) were examined by immuno-histochemistry; in 62% of cases core or incisional biopsies were taken before drug administration, allowing comparison in paired biopsies of the cytological and molecular changes induced by treatment Sixty percent of the patients received relatively high doses of FAC or FEC (5-fluorouracil, doxorubicin or epirubicin and cyclophosphamide), and 40% received relatively high doses of doxorubicin or epirubicin alone. No significant changes were observed in the molecular markers studied following chemotherapy; in the few biopsies where changes appeared, the changes did not exhibit any significant or similar trend. For 30 of the patients who received FAC/FEC treatment, follow-up reached a median of 34 months. In these cases, neither the clinical (reduction in tumor size) nor the histological (evaluated after neoadjuvant chemotherapy) responses showed statistically significant differences between the patients who developed distant metastases and the disease-free patients. c-erbB-2 was over-expressed in 50% of patients who developed distant metastases vs. 7% of the disease-free patients. Disease free survival (DFS) curves between c-erbB-2-positive and c-erbB-2-negative patients were statistically significant. No correlation between p53 or P170 expression with DFS was found. Our results suggest that c-erbB-2 protein expression is associated with development of distant metastases in breast cancer patients treated with relatively high doses of anthracyclines in induction chemotherapy.

Solution structure determination by two-dimensional 1H NMR of omega-conotoxin MVIID, a calcium channel blocker peptide.

The three-dimensional structure of omega-conotoxin MVIID has been determined in aqueous solution by two-dimensional 1H NMR techniques. A total of 267 relevant upper-bound distance restraints were used to obtain a family of convergent structures using molecular dynamics methods. A standard simulated annealing protocol using the XPLOR program included in ARIA provided a total of 18 final structures. The averaged RMSD between these structures and the mean atomic coordinates was 0.8 +/- 0.3 A for the backbone atoms. The highest mobility was observed in the segments between residues 10 to 13, comprising Tyr 13, one of the residues shown to be important for binding of omega-conotoxin GVIA and MVIIA to N-type calcium channels. The three-dimensional structure is stabilised by the three disulfide bonds and includes a short antiparallel beta-strand between residues 5-8, 23-25 and 19-21. The folding for this non-N-type calcium channel blocker is similar to that previously calculated for omega-conotoxins GVIA, MVIIA and MVIIC. This suggests the disulfide bond pattern fixes the structure. The reported three-dimensional information can be used to advantage in order to highlight the structural parameters involved in discrimination among calcium channel subtypes.

Heat shock protein expression and drug resistance in breast cancer patients treated with induction chemotherapy.

Heat shock proteins (Hsps) are induced in vitro by several cytotoxic drugs; in human breast cancer cells these proteins appear to be involved in anti-cancer drug resistance. The present report was designed to analyze whether chemotherapy affects in vivo the expression of Hsp27, Hsp70, Hsc70 and Hsp90 in breast cancer patients treated with induction chemotherapy and whether these proteins may be determinants of tumor resistance to drug administration. We have analyzed 35 biopsies from breast cancer patients treated with induction chemotherapy. Expression of the Hsps in the tumors was compared with (i) histological and clinical responses to chemotherapy, (ii) tumor cell proliferation measured by proliferating cell nuclear antigen (PCNA) immunostaining and nucleolar organizer regions (AgNORs) staining and (iii) the expression of estrogen and progesterone receptors. We also compared disease-free survival (DFS) and overall survival (OS) with the expression of the Hsps studied. After chemotherapy, nuclear Hsp27 and Hsp70 expression was increased and Hsp70 and Hsc70 cytoplasmic expression was decreased. A high nuclear proportion of Hsp70 in tumor cells (>10%) correlated significantly with drug resistance. We also observed that patients whose tumors expressed nuclear or a high cytoplasmic proportion (>66%) of Hsp27 had shorter DFS. The combination of Hsp27 and Hsp70 levels showed a strong correlation with DFS. Neither the cellular proliferation nor the levels of steroid receptors showed any significant difference before or after drug administration or during follow-up of patients. Our results suggest that Hsp27 and Hsp70 are involved in drug resistance in breast cancer patients treated with combination chemotherapies.

Integration of estrogen and progesterone receptors with pathological and molecular prognostic factors in breast cancer patients.

In this study we have examined biopsies from women with localized primary breast cancer to investigate the prognostic performance of estrogen receptors (ER) and progesterone receptors (PR) for estimating the metastatic probability of the patients, and to explore whether discrimination gets better by combining clinicopathological and other molecular parameters into a score. This prospective study involved 205 patients with a median follow-up of 5 y. Among the evaluated clinicopathological data were: patient's age; tumor size; axillary lymph node involvement; and tumor grade. The most representative tumor samples were derived to a single laboratory for immunohistochemical evaluation of the following molecular markers: ER, PR, proliferating cell nuclear antigen (PCNA), p53 protein product, erbB-2 (HER-2/neu) oncoprotein, and P170 glycoprotein (mdrl gen product). Distant metastases (study endpoint) appeared in 19.5% (40/205) of the patients, most of these patients presented a mixture of poor, regular and good prognostic factors. Disease-free survival analysis procedures (Kaplan-Meier method) identified tumor size, axillary lymph node involvement, tumor grade, receptor status, PCNA, p53, erbB-2 and P170 as useful prognostic factors. Proportional hazard regression analysis (Cox) identified in order of importance erbB-2, tumor size, receptors status, tumor grade and PCNA as useful prognostic factors. To facilitate the evaluation of the prognostic factors, a practical and simple score system was derived. A high pathological score identified 65% of the patients that developed distant metastases, while a high molecular score was obtained in 57% of patients with metastatic disease. There was a significant improvement in the diagnosis of probability of being with distant metastases when the pathological score was combined with the molecular score, 82% of the patients with distant metastases showed an elevated combined score. Validation of this scoring system will need further larger studies (validation set as opposed to the training set used in the present study). Due to the complexity of events in cancer, the evaluation of a combination of prognostic factors should be of value to clinicians to make a more objective estimate of the prognosis of individual breast cancer patients.

Structure-affinity relationships for the binding of actinomycin D to DNA.

Molecular models of the complexes between actinomycin D and 14 different DNA hexamers were built based on the X-ray crystal structure of the actinomycin-d(GAAGCTTC)2 complex. The DNA sequences included the canonical GpC binding step flanked by different base pairs, nonclassical binding sites such as GpG and GpT, and sites containing 2,6-diamino-purine. A good correlation was found between the intermolecular interaction energies calculated for the refined complexes and the relative preferences of actinomycin binding to standard and modified DNA. A detailed energy decomposition into van der Waals and electrostatic components for the interactions between the DNA base pairs and either the chromophore or the peptidic part of the antibiotic was performed for each complex. The resulting energy matrix was then subjected to principal component analysis, which showed that actinomycin D discriminates among different DNA sequences by an interplay of hydrogen bonding and stacking interactions. The structure-affinity relationships for this important antitumor drug are thus rationalized and may be used to advantage in design of novel sequence-specific DNA-binding agents.

Heat shock proteins and cell proliferation in human breast cancer biopsy samples.

Human breast cancers may overexpress certain heat shock protein (hsp) family members, proteins which are involved with cell proliferation and differentiation as well as with disease prognosis and drug resistance. Here, we have studied the relationship between the expression of two hsps (hsp27 and hsp70) and the proliferative activity of tumor cells in 40 biopsies from breast cancer patients. Twenty of these tumors were selected for a detailed colocalization study. Immunocytochemistry was done using specific antibodies against hsp27 and hsp70. Cell proliferation was studied analyzing the expression of proliferating cell nuclear antigen (PCNA) (late G1, S, and G2 phases of the cell cycle) and the number of silver-staining nucleolar organizer regions (AgNORs) (G1 phase). The colocalization study revealed a statistically significant inverse correlation between hsp27 expression and cell proliferation in 16/19 (84%) of the cases evaluated by PCNA immunostaining, and in 11/16 (69%) of the cases evaluated by AgNORs. In contrast, a statistically significant positive correlation between hsp70 expression and elevated cell proliferation was seen in almost 85% of the cases evaluated by PCNA staining, and in almost 50% of the cases evaluated by AgNORs. Moreover, in 22% (9/40) of the breast cancer samples examined, hsp70 was clearly associated with the mitotic spindle. A Western blot analysis revealed that hsp70 was coprecipitated with taxol-polymerized tubulin. The association of hsp70 with the mitotic spindle was not clearly noted in lung carcinoma samples (N = 20) or in normal cells displaying elevated mitotic activity. These studies thus demonstrate that in a significant percentage of clinical breast cancers hsp27 overexpression is inversely correlated with cell proliferation, while hsp70 is clearly associated with the mitotic spindle and cell proliferation. These results add evidence to the concept that in human breast cancers hsp27 may be involved in cell growth arrest and increased differentiation while, in contrast, hsp70 may be involved in cell proliferation; further studies will be necessary to elucidate these possible cause-and-effect relationships.