John S. Sussenbach

Mitogenic Signaling of Insulin-like Growth Factor I in MCF-7 Human Breast Cancer Cells Requires Phosphatidylinositol 3-Kinase and Is Independent of Mitogen-activated Protein Kinase

Addition of insulin-like growth factor I (IGF-I) to quiescent breast tumor-derived MCF-7 cells causes stimulation of cyclin D1 synthesis, hyperphosphorylation of the retinoblastoma protein pRb, DNA synthesis, and cell division. All of these effects are independent of the mitogen-activated protein kinase (MAPK) pathway since none of them is blocked by PD098059, the specific inhibitor of the MAPK activating kinase MEK1. This observation is consistent with the finding that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a strong inducer of MAPK activity in MCF-7 cells, effectively inhibits proliferation. The anti-proliferative effect of TPA in these cells may be accounted for, at least in part, by the MAPK-dependent stimulation of the synthesis of p21WAF1/CIP1, an inhibitor of cyclin/cyclin-dependent kinase complexes. In contrast, all of the observed stimulatory effects of IGF-I on cell cycle progression, cyclin D1 synthesis, and pRb hyperphosphorylation were blocked by the specific phosphatidylinositol 3-kinase inhibitor LY294002, suggesting that phosphatidylinositol 3-kinase activity but not MAPK activity is required for transduction of the mitogenic IGF-I signal in MCF-7 cells.

The human insulin-like growth factor II gene contains two development-specific promoters

The insulin‐like growth factors (IGF) play an important role in fetal and postnatal development. Recently, the nucleotide sequences of the cDNAs encoding IGF‐I and IGF‐II and part of the human IGF genes were reported. In this communication we describe two distinct IGF‐II cDNAs isolated from a human adult liver and a human hepatoma cDNA library, respectively. Using these two cDNAs, we have established that the human IGF‐II gene contains at least 7 exons. Two different IGF‐II promoters have been identified, 19 kilobases (kb) apart, which are active in a development‐specific manner. The promoter, active in the adult stage, is located only 1.4 kb downstream from the insulin gene.

Nucleotide sequences of cDNAs encoding precursors of human insulin-like growth factor II (IGF-II) and an IGF-II variant

We have isolated 3 cDNA clones encoding human IGF‐II and a variant of IGF‐II. The amino acid sequence encoded by the IGF‐II cDNA is identical to the sequence previously described [(1978) FEBS Lett. 89, 283‐286]. In the amino acid sequence predicted by the IGF‐II variant cDNA, the Ser residue 29 in the B‐domain has been replaced by an Arg‐Leu‐Pro‐Gly sequence. The corresponding mRNAs probably arise by alternative splicing of a common RNA precursor. The IGF coding region of the cDNA inserts is flanked by sequences encoding a signal peptide and a carboxy‐terminal peptide indicating that both human IGF‐II and its variant are synthesized as precursors.

Initial characterization of the four promoters of the human insulin-like growth factor II gene

The human insulin-like growth factor II (IGF-II) gene contains four promoters (P1-P4), which are expressed in a tissue-specific and development-dependent way. Analysis of IGF-II mRNAs in different tissues has revealed that promoters P3 and P4 are expressed in all fetal and in nonhepatic adult tissues. In adult liver, however, the promoters P2, P3 and P4 are completely shut off and another promoter, P1, is activated. To obtain more insight in the mechanisms involved in the regulation of IGF-II gene expression we have performed an initial characterization of the IGF-II promoters employing transient expression of IGF-II promoter constructs in Hep3B and HeLa cells. These studies have revealed that promoters P1, P3 and P4 are active in both cell lines tested, while no activity of promoter P2 could be detected. Employing gel retardation and DNaseI footprint analysis we have identified in the three IGF-II promoters a number of elements which are bound by nuclear proteins.

The gene structure of the insulin-like growth factor family

The insulin-like growth factors (IGF) constitute a family of proteins with insulin-like and growth-stimulating properties. The best characterized members of this family are IGF-I, a protein of 70 amino acids which plays an important role in post-natal growth, and IGF-II, a 67 amino acid protein which is most likely involved in fetal development. The gene structure of IGF-II has been elucidated for the human and the rat and shows extensive interspecies homologies. The gene structure of IGF-I has only partially been established. A striking feature of the IGF genes is that they are controlled by multiple promoters which are expressed in a tissue-specific and development-dependent way.

Expression of insulin-like growth factor-I and -II genes in human smooth muscle tumours.

The insulin‐like growth factors I and II (IGF‐I and ‐II) are polypeptides which play an important role in growth and development of the organism. In the present report we describe the detection of human IGF‐I RNAs (both type Ia and type Ib) and IGF‐II RNAs in benign (leiomyoma) and malignant (leiomyosarcoma) tumours from smooth muscle origin, using Northern blot hybridization analysis. In normal smooth muscle tissue of the uterus we found low levels of IGF‐I RNAs only. In the tumours the same IGF‐I RNA species were detected as in adult non‐tumour tissues (uterus, liver). For transcription of the IGF‐II gene in these tumours, two promoters are used which are expressed in fetal liver, but not in adult liver. The presence of IGF‐I and IGF‐II RNAs was also established by nucleotide sequence analysis of recombinant DNA clones isolated from cDNA libraries derived from two leiomyosarcomas. The nucleotide sequences of these cDNA clones, together covering the entire coding regions of IGF‐Ia and IGF‐II var RNA, predict that IGFs encoded by the tumour RNAs do not differ in amino acid sequence from the corresponding polypeptides isolated from serum. In those tissues containing IGF‐I RNAs, IGF‐I immunoreactivity was also demonstrated.

The role of MAP kinase in TPA-mediated cell cycle arrest of human breast cancer cells

In MCF7 breast cancer cells, mitogen-activated protein (MAP) kinase (i.e. Erk-1/2) is activated by the mitogen insulin, but also by the growth inhibiting agent TPA, though with very different kinetics. Insulin induces a relatively transient activation of Erk2 (<15 min), whereas TPA is able to induce a prolonged activation of Erk2 (>6 h). Expression of immediate-early genes of the c-fos and c-jun families, whose transcription and activation are regulated by MAP kinases, is differentially induced by insulin and TPA. Whereas insulin stimulates prolonged induction of c-jun, but not of junB mRNA, resulting in c-jun expression during the entire G1 period, the growth inhibitor TPA induces junB much longer than c-jun. Inhibition of the Erk2 pathway by PD98059, specific for the upstream MAP kinase kinase (MEK1), abolishes TPA-stimulated junB but not insulin-induced c-jun. In agreement with this, insulin readily stimulates Jun kinase (JNK), whereas TPA does not. Furthermore, insulin-induced pRB hyperphosphorylation at the G1-S transition and S-phase entry is insensitive to MAP kinase inhibition by PD98059. On the other hand, PD98059 reverts the inhibitory effect of TPA on cell cycle entry as well as on pRB hyperphosphorylation, indicating that Erk effectors function as inhibitors of proliferation in MCF7 cells.

An Adenovirus Type 5 Gene Function Required for Initiation of Viral DNA Replication

Adenovirus type 5 (Ad5) DNA replication was studied after infection of human or monkey cells with two DNA-negative temperature-sensitive mutants belonging to different complementation groups (H5ts125 and H5ts36). When infection was carried out at the permissive temperature (32°) followed by a shift to the nonpermissive temperature (39.5°) viral DNA synthesis in H5ts125-infected cells was reduced 90% within 1 hr after shift-up, while a decline in DNA synthesis in H5ts36-infected cells is only observed after 6 hr. Analysis of the various forms of DNA synthesized under conditions of inhibition showed a constant ratio of replicating to mature viral DNA for both mutants, while no accumulation of replicating molecules was observed. When H5ts125-infected cells were pulse-labeled with [3H]thymidine at 32 or 39.5° followed by a chase of the label at 39.5°, replicating DNA was converted into mature DNA at the same rate as in wild-type-infected cells. This indicates that chain propagation and termination could occur normally under nonpermissive conditions. The results of density labeling experiments performed at 39.5° are in agreement with an initiation block in H5ts125-infected cells at the nonpermissive temperature. It is concluded that the H5ts125 gene product and possibly also the H5ts36 gene product are required for the initiation of new rounds of replication. The potential role in initiation of the adenovirus-specific DNA binding protein, which is coded for by the H5ts125 gene, is discussed.

Organization of the human genes for insulin-like growth factors I and II

Recently, we have reported the isolation of cDNAs encoding the precursors of insulin‐like growth factors I and II (IGF‐I and II) [(1983) Nature 306, 609‐611; (1985) FEBS Lett. 179, 243‐246. These cDNAs were employed as specific probes to detect and isolate the corresponding genes from human cosmid DNA libraries. Three cosmids were detected, together containing the entire cDNA sequence of IGF‐I, and one cosmid containing the sequence of IGF‐II cDNA. Southern blot hybridization, physical mapping and nucleotide sequence analysis of these cosmids revealed that the IGF‐I and ‐II genes have a discontinous structure. The IGF‐I gene contains at least four exons spanning a region of probably more that 45 kilobasepairs (kb), while the IGF‐II gene consists of at least five exons, spanning a region of 16 kb.

Identification of multiple transcription start sites in the human insulin-like growth factor-I gene.

We have localized four transcription initiation sites in the human insulin-like growth factor-I (IGF-I) gene. Two transcription start sites were identified which result in a longer and shorter version of the leader derived from the known exon 1 of the IGF-I gene. Transcription starting at the upstream transcription initiation site results in a leader exon 1 of about 1155 nucleotides (nt), whereas transcription starting at the downstream initiation site results in a leader of about 240 nt. The majority of the transcripts initiate at the latter site. We further identified a region in the human IGF-I gene between exons 1 and 2, which shows a high degree of homology with the rat IGF-I leader exon 1B. By means of the polymerase chain reaction (PCR) we detected human IGF-I mRNAs containing this novel leader. The corresponding exon was designated exon 1B according to the rat IGF-I gene terminology. PCR and RNase protection analyses identified two transcription start sites within this alternative leader exon 1B. Transcription initiated at the most upstream start site results in a leader of about 750 nt, whereas transcription starting at the downstream site is heterogeneous, resulting in leaders of 65-75 nt long. No consensus TATA-box or AT-rich regions are present immediately upstream of all four transcription start sites identified, nor are these regions particularly GC-rich. The IGF-I gene is known to be expressed differentially in a tissue- and development-specific fashion. Differential activation of multiple promoters could very well play a crucial role in IGF-I gene regulation.