Background The HER2 gene, which encodes the growth factor receptor HER2, is amplified and HER2 is overexpressed in 25 to 30 percent of breast cancers, increasing the aggressiveness of the tumor. Methods We evaluated the efficacy and safety of trastuzumab, a recombinant monoclonal antibody against HER2, in women with metastatic breast cancer that overexpressed HER2. We randomly assigned 234 patients to receive standard chemotherapy alone and 235 patients to receive standard chemotherapy plus trastuzumab. Patients who had not previously received adjuvant (postoperative) therapy with an anthracycline were treated with doxorubicin (or epirubicin in the case of 36 women) and cyclophosphamide with (143 women) or without trastuzumab (138 women). Patients who had previously received adjuvant anthracycline were treated with paclitaxel alone (96 women) or paclitaxel with trastuzumab (92 women). Results The addition of trastuzumab to chemotherapy was associated with a longer time to disease progression (median, 7.4 ...

Use of Chemotherapy plus a Monoclonal Antibody against HER2 for Metastatic Breast Cancer That Overexpresses HER2

We have used the Escherichia coli beta-glucuronidase gene (GUS) as a gene fusion marker for analysis of gene expression in transformed plants. Higher plants tested lack intrinsic beta-glucuronidase activity, thus enhancing the sensitivity with which measurements can be made. We have constructed gene fusions using the cauliflower mosaic virus (CaMV) 35S promoter or the promoter from a gene encoding the small subunit of ribulose bisphosphate carboxylase (rbcS) to direct the expression of beta-glucuronidase in transformed plants. Expression of GUS can be measured accurately using fluorometric assays of very small amounts of transformed plant tissue. Plants expressing GUS are normal, healthy and fertile. GUS is very stable, and tissue extracts continue to show high levels of GUS activity after prolonged storage. Histochemical analysis has been used to demonstrate the localization of gene activity in cells and tissues of transformed plants.

/pdf/gus-fusions-beta-glucuronidase-as-a-sensitive-and-versatile-4b3n5r6gfw.pdf

GUS fusions: beta‐glucuronidase as a sensitive and versatile gene fusion marker in higher plants.

Analyses of steroid receptors are important for understanding molecular details of transcriptional control, as well as providing insight as to how an individual transacting factor contributes to cell identity and function. These studies have led to the identification of a superfamily of regulatory proteins that include receptors for thyroid hormone and the vertebrate morphogen retinoic acid. Although animals employ complex and often distinct ways to control their physiology and development, the discovery of receptor-related molecules in a wide range of species suggests that mechanisms underlying morphogenesis and homeostasis may be more ubiquitous than previously expected.

/pdf/the-steroid-and-thyroid-hormone-receptor-superfamily-1r0qhjfpv5.pdf

The steroid and thyroid hormone receptor superfamily

A tandem repeat in DNA is two or more contiguous, approximate copies of a pattern of nucleotides. Tandem repeats have been shown to cause human disease, may play a variety of regulatory and evolutionary roles and are important laboratory and analytic tools. Extensive knowledge about pattern size, copy number, mutational history, etc. for tandem repeats has been limited by the inability to easily detect them in genomic sequence data. In this paper, we present a new algorithm for finding tandem repeats which works without the need to specify either the pattern or pattern size. We model tandem repeats by percent identity and frequency of indels between adjacent pattern copies and use statistically based recognition criteria. We demonstrate the algorithm’s speed and its ability to detect tandem repeats that have undergone extensive mutational change by analyzing four sequences: the human frataxin gene, the human β T cell receptor locus sequence and two yeast chromosomes. These sequences range in size from 3 kb up to 700 kb. A World Wide Web server interface at c3.biomath.mssm.edu/trf.html has been established for automated use of the program.

/pdf/tandem-repeats-finder-a-program-to-analyze-dna-sequences-1oo3g6vnch.pdf

Tandem repeats finder: a program to analyze DNA sequences

When epidermal growth factor and its relatives bind the ErbB family of receptors, they trigger a rich network of signalling pathways, culminating in responses ranging from cell division to death, motility to adhesion. The network is often dysregulated in cancer and lends credence to the mantra that molecular understanding yields clinical benefit: over 25,000 women with breast cancer have now been treated with trastuzumab (Herceptin), a recombinant antibody designed to block the receptor ErbB2. Likewise, small-molecule enzyme inhibitors and monoclonal antibodies to ErbB1 are in advanced phases of clinical testing. What can this pathway teach us about translating basic science into clinical use?

/pdf/untangling-the-erbb-signalling-network-1b92otnrud.pdf

Untangling the ErbB signalling network

We constructed a series of recombinant genomes which directed expression of the enzyme chloramphenicol acetyltransferase (CAT) in mammalian cells. The prototype recombinant in this series, pSV2-cat, consisted of the beta-lactamase gene and origin of replication from pBR322 coupled to a simian virus 40 (SV40) early transcription region into which CAT coding sequences were inserted. Readily measured levels of CAT accumulated within 48 h after the introduction of pSV2-cat DNA into African green monkey kidney CV-1 cells. Because endogenous CAT activity is not present in CV-1 or other mammalian cells, and because rapid, sensitive assays for CAT activity are available, these recombinants provided a uniquely convenient system for monitoring the expression of foreign DNAs in tissue culture cells. To demonstrate the usefulness of this system, we constructed derivatives of pSV2-cat from which part or all of the SV40 promoter region was removed. Deletion of one copy of the 72-base-pair repeat sequence in the SV40 promoter caused no significant decrease in CAT synthesis in monkey kidney CV-1 cells; however, an additional deletion of 50 base pairs from the second copy of the repeats reduced CAT synthesis to 11% of its level in the wild type. We also constructed a recombinant, pSV0-cat, in which the entire SV40 promoter region was removed and a unique HindIII site was substituted for the insertion of other promoter sequences.

Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells.

The murine monoclonal antibody mumAb4D5, directed against human epidermal growth factor receptor 2 (p185HER2), specifically inhibits proliferation of human tumor cells overexpressing p185HER2. However, the efficacy of mumAb4D5 in human cancer therapy is likely to be limited by a human anti-mouse antibody response and lack of effector functions. A "humanized" antibody, humAb4D5-1, containing only the antigen binding loops from mumAb4D5 and human variable region framework residues plus IgG1 constant domains was constructed. Light- and heavy-chain variable regions were simultaneously humanized in one step by "gene conversion mutagenesis" using 311-mer and 361-mer preassembled oligonucleotides, respectively. The humAb4D5-1 variant does not block the proliferation of human breast carcinoma SK-BR-3 cells, which overexpress p185HER2, despite tight antigen binding (Kd = 25 nM). One of seven additional humanized variants designed by molecular modeling (humAb4D5-8) binds the p185HER2 antigen 250-fold and 3-fold more tightly than humAb4D5-1 and mumAb4D5, respectively. In addition, humAb4D5-8 has potency comparable to the murine antibody in blocking SK-BR-3 cell proliferation. Furthermore, humAb4D5-8 is much more efficient in supporting antibody-dependent cellular cytotoxicity against SK-BR-3 cells than mumAb4D5, but it does not efficiently kill WI-38 cells, which express p185HER2 at lower levels.

https://www.pnas.org/content/pnas/89/10/4285.full.pdf

Humanization of an anti-p185HER2 antibody for human cancer therapy.

We characterized the transcriptional activity of the long terminal repeat (LTR) of Rous sarcoma virus by constructing a recombinant plasmid, pRSVcat, in which bacterial chloramphenicol acetyltransferase (CAT; acetyl-CoA:chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28) coding sequences are placed under LTR control. We find that the LTR directs relatively high levels of CAT synthesis within 48 hr after calcium phosphate-mediated introduction of this plasmid into CV-1 monkey kidney cells, chicken embryo fibroblasts, Chinese hamster ovary cells, HeLa cells, or mouse NIH/3T3 cells. The level of CAT synthesis is 3-fold higher in CV-1 cells and up to 10-fold higher in HeLa and mouse NIH/3T3 cells than after transfection with a related vector, pSV2cat, carrying CAT sequences under control of the simian virus 40 early promoter. We have shown, by primer extension, that the amounts of CAT-specific mRNAs encoded by pRSVcat and pSV2cat correlate with the levels of CAT enzyme activity. By both S1 nuclease mapping and primer extension, we have demonstrated that the start site for RNA transcription within the LTR of pRSVcat corresponds to previous mapping data. We estimated transfection efficiencies by monitoring immunofluorescence induced by a rhodamine-labeled CAT antibody. Our results indicate that the Rous sarcoma virus LTR can direct synthesis of high levels of functional mRNA and has a wide expression range. The observed high transcriptional activity of the LTR is significant because it has been postulated that this LTR promotes activity of adjacent cellular oncogenes.

https://www.pnas.org/content/pnas/79/22/6777.full.pdf

The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by DNA-mediated transfection

IgE antibodies bind to specific high-affinity receptors on mast cells, leading to mast cell degranulation and release of mediators, such as histamine, which produce symptoms associated with allergy. Hence, anti-IgE antibodies that block binding of IgE to its high-affinity receptor are of potential therapeutic value in the treatment of allergy. These antibodies must also not bind to IgE once it is bound to the receptor because this would trigger histamine release. This study describes the humanization of a murine antibody, MaE11, with these characteristics. Variants of the humanized antibody were evaluated to probe the importance of framework residues on antibody binding and to determine which charged residues in the CDR interacted with IgE. We found that only five changes in human framework residues were required to provide for binding comparable to that of the original murine antibody.

Humanization of an antibody directed against IgE.

Growth of human breast cells is closely regulated by steroid hormone as well as peptide hormone receptors. Members of both receptor classes are important prognostic factors in human breast cancer. Clinical data indicate that overexpression of the HER-2 gene is associated with an estrogen receptor-negative phenotype. In this study, we demonstrate that introduction of a HER-2 cDNA, converting non-overexpressing breast cancer cells to those which overexpress this receptor, results in development of estrogen-independent growth which is insensitive to both estrogen and the antiestrogen, tamoxifen. Moreover, activation of the HER-2 receptor in breast cancer cells by the peptide growth factor, heregulin, leads to direct and rapid phosphorylation of ER on tyrosine residues. This is followed by interaction between ER and the estrogen-response elements in the nucleus and production of an estrogen-induced protein, progesterone receptor. In addition, overexpression of HER-2 receptor in estrogen-dependent tumor cells promotes ligand-independent down-regulation of ER and a delayed autoregulatory suppression of ER transcripts. These data demonstrate a direct link between these two receptor pathways and suggest one mechanism for development of endocrine resistance in human breast cancers.

Cornelia M. Gorman

Papers

Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells.

Humanization of an anti-p185HER2 antibody for human cancer therapy.

The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by DNA-mediated transfection

Humanization of an antibody directed against IgE.

HER-2 tyrosine kinase pathway targets estrogen receptor and promotes hormone-independent growth in human breast cancer cells.