Compromiso

3.2.1 Myelinating cultures

By infecting rat Schwann cells with retroviruses carrying rat Pmp22 D’Urso et al. (1997) produced Schwann cell lines which over or underexpressed Pmp22. Pmp22

overexpressing (sense construct), underexpressing (antisense construct) and control Schwann cells were co-cultured with purified DRG neurons. Pmp22 overexpression reduced the proliferation rate of Schwann cells by 60% under growth conditions in a medium which supported Schwann cell proliferation but did not allow basal lamina formation and myelination. When cultures were switched to medium which promoted myelination, Pmp22 expression gradually increased in all cultures (D'Urso et al. 1997). Pmp22 mRNA levels were increased in Schwann cells expressing the sense construct and slightly decreased in cells expressing the antisense construct when compared with control cultures. PMP22 immunoreactive fibres first appeared 7 d after the addition of ascorbate to the medium in all three culture types. Schwann cells in all cultures estabhshed contacts with axons, ensheathed them and formed myelin which looked normal when analysed by electron microscopy. No morphological differences were observed between control and recombinant cell- cultures in either proliferation or myelination medium. Overexpression of Pmp22

did not affect either the onset of myelination or the expression of other myelin genes (D’Urso et al. 1997). Both Po and MBP immunoreactivity appeared synchronously with PMP22 and was correctly targeted to myelin membranes. PMP22 immunostaining was seen not only in the myelin sheath but also within the

cytoplasm of myelinating Schwann cells in overexpressing cultures (D'Urso et al. 1997). In contrast Po and MBP were localised only in compact myelin. On this basis the authors speculated that PMP22 may perform different functions in different cellular compartments during myelination. Only myelinating Schwann cells were able to target PMP22 to the plasma membrane, non-myelinating cells in overexpressing cocultures had detectable levels of PMP22 but the signal was only present in the perinuclear compartments. The authors concluded that PMP22 was not one of the key molecules involved in the early spiralling events that occur soon after a 1:1 relationship between Schwann cell and axon have been established but is more involved in controlling myelin thickness.

3.2.2. Cultured Schwann cells.

Altered Pmp22 levels significantly influenced DNA synthesis in cultured Schwann cells. In Pmp22 overexpressing Schwann cells the level of DNA synthesis dropped to 60% of control values; conversely underexpression correlated with enhanced DNA levels (150% of control values) (Zoidl et al. 1995). Both the CD25 and the SRI3 PMP22 mRNA transcripts increased as Schwann cells stopped proliferating and both were rapidly downregulated as they re-entered the cell cycle. Altered levels of Pmp22 expression also altered the entry of quiescent cells into the cell cycle. Overexpression of Pmp22

delayed serum and forskolin stimulated entry of resting cells from GO/Gl to the S+G2/M phase by 8 h. Cells expressing reduced Pmp22 levels did not enter the cell cycle faster than controls, but the proportion of cells that re-entered the cell cycle was consistently higher. This suggests that low levels of Pmp22 expression may facilitate the transition from GO/Gl to S phase for an increased number of resting cells.

The growth of Schwann cells purified from 3 d old Pmp22 overexpressing rats (Sereda et al. 1996) showed growth characteristics very similar to control cultures.

Pmp22 transgenic Schwann cells showed no signs of premature growth arrest, and

no evidence o f increased apoptotic death.

3.2.3 Fibroblasts.

Cultured fibroblasts overexpressing PMP22/Pmp22 showed morphological hallmarks usually associated with programmed cell death (Fabbretti et al. 1995; Zoidl et al. 1997). These included a collapsed cellular body and condensed nuclei accompanied by cell rounding and membrane blebbing. Nuclear morphology was altered in 54% of PMP22 overexpressing fibroblasts (Fabbretti et al. 1995). When transfected with cDNA constructs for PMP22 point mutations (L16P, S79C, T118M ) the point mutations showed a significantly decreased ability

to induce nuclear condensation and expressed normal nuclear morphology (Fabbretti et al. 1995). This decreased ability to induce morphological alterations was also found in fibroblasts overexpressing the Trembler alteration (Zoidl et al. 1997). They found that the proportion of fi-agmented nuclei increased during the culture period in PMP22 (22% to 61%) and to a lesser extent PMP22^^*^°^ (12%- 22%) overexpressing cells. This suggests that PMP22 acts in a physiological manner because intact PMP22 but not the mutant Tr protein promotes increased cell degeneration.

The lack of the apoptotic-like phenotype in REF-52 cells, which do not normally express PMP22, suggests a cell type specific response (Fabbretti et al. 1995). From this evidence Fabretti et al. concluded that PMP22 itself does not induce apoptosis but permits entry into a state in which apoptosis becomes more accessible (Fabbretti et al. 1995).

Elevated levels of PMP22 and PMP22^^*^®'^ significantly reduced fibroblast proliferation with PMP22®^^^®'^ having a less marked effect. An accumulation of cells in the G1 compartment of the cell cycle indicates that elevated expression levels prevent NIH3T3 fibroblasts from undergoing DNA replication (Zoidl et al.

1997).

3.2.4 Yeast cells.

When human PMP22 cDNAs were cloned into yeast cells northern blots showed a strong signal, while western blots did not. This was taken to mean that the PMP22 protein was degraded rapidly in yeast cells to protect the cells from its toxic effects. These toxic effects are implied by a decreased growth rate in PMP22 induced yeast cells. In contrast, following amplification of the PMP22 gene in two human tumour cell lines, PMP22 was detected by western blot (Park et al. 1997).

In document Análisis del discurso en la construcción de la identidad urbana contemporánea: estudio de caso en la Heroica Puebla de Zaragoza, Puebla (página 156-164)